Human kynureninase enzymes and uses thereof

ABSTRACT

Methods and compositions related to the use of a protein with kynureninase activity are described. For example, in certain aspects there may be disclosed a modified kynureninase capable of degrading kynurenine. Furthermore, certain aspects of the invention provide compositions and methods for the treatment of cancer with kynurenine depletion using the disclosed proteins or nucleic acids.

This application claims the benefit of U.S. Provisional PatentApplication No. 62/658,261, filed Apr. 16, 2018, the entirety of whichis incorporated herein by reference.

This invention was made with government support under Grant No. R01CA154754 awarded by the National Institutes of Health. The U.S.government has certain rights in the invention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the field of molecularbiology and oncology. More particularly, it concerns engineeredkynureninase enzymes and methods for using the same.

2. Description of Related Art

Kynurenine is a metabolite of the amino acid Tryptophan generated viathe action of either indolamine 2,3 dioxygenase (IDO) or tryptophan 2,3dioxygenase (TDO). Kynurenine exerts multiple effects on cellphysiology; one of the most important of which is as a modulator of Tcell responses. Many tumor cells regulate the synthesis of IDO and/orTDO enzymes to elevate the local concentration of Kynurenine which isaccompanied with depletion of Tryptophan. High levels of Kynurenine, inturn serve as a powerful way to inhibit the function of tumorinfiltrating T cells that would otherwise attack the tumor.

The significance of Kynurenine production in cancer is well recognizedand has led to the development of inhibitors of theKynurenine-generating enzymes IDO and TDO; at least one of thesecompounds are currently undergoing Phase II clinical evaluation. HoweverIDO or TDO inhibition is problematic for cancer therapy because: (1)there are two isoforms of IDO and together with TDO, the inhibition ofall possible pathways for Kynurenine generation at present requires thegeneration of multiple small molecule inhibitors; (2) resistance toinhibition can arise.

The human Kynureninase has a strong preference for the degradation of3′OH Kynurenine which is degraded with a catalytic efficiency (Kcat/Km)about a 1,000 higher than that displayed for Kynurenine degradation. Incontrast some bacterial enzymes such as the enzyme from P. fluorescenshave strong preference for Kynurenine over 3′OH Kynurenine. Fortherapeutic purposes it is essential to have an agent (i.e. atherapeutic enzyme) that has the requisite pharmacological propertiesfor in vivo applications in humans. These include: (i) high catalyticactivity for Kynurenine, (ii) enhanced stability to deactivation inserum in order to achieve prolonged depletion of Kynurenine in serum andtissues upon single dose injection

SUMMARY OF THE INVENTION

Aspects of the present invention overcome a major deficiency in the artby providing enzymes that comprise engineered polypeptide sequencescapable of degrading L-kynurenine, having a high degree of sequenceidentity with the human enzyme to avoid the elicitation of adverseimmune responses and displaying favorable pharmacokinetics in serum asdesired for cancer therapy. In PCT/US2014/053437, the inventorsdisclosed mutant human kynureninase (also referred to herein as h-KYNaseor HsKYN) enzymes having up to 24-fold higher catalytic efficiency forthe degradation of KYN relative to the wild-type human enzyme. There isa need for the discovery of mutant h-KYNase enzymes having highercatalytic activity and stability in human serum relative to thosedisclosed in PCT/US2014/053437. Aspects of the present applicationaddresses this need and discloses mutant enzymes having higher catalyticefficiencies and also mutant enzymes having higher catalytic activitythan the wild type h-KYNase as well as enzymes with enhanced serumstability.

In a first embodiment, the invention provides an isolated, modifiedhuman kynureninase enzyme, said modified enzyme having an amino acidsequence least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a humankynureninase enzyme of SEQ ID NO: 1, 2, 3 or one of the polypeptidesprovided in Table 1 and comprising at least one substitution (relativeto sequence of SEQ ID NO: 1) selected from the group consisting of L8P,K38E, Y47L, I48F, K50Q, I51M, S60N, K64N, D65G, E66K, N67D, N67P, D67S,A68F, A68T, A68V, F71L, F71M, L72N, K84E, E88N, E89K, E89S, E90Q, D92E,K93N, K93T, A95H, A95Q, K96N, I97H, I97L, I97V, A98G, A99G, A99I, A99R,A99S, A99T, A99V, Y100N, Y100S, Y100T, G101A, H102W, E103F, E103H,E103N, E103Q, E103R, E103V, E103W, V104D, V104E, V104F, V104H, V104K,V104L, V104R, G105A, G105H, G105S, G105T, E106D, K106D, K106E, K106H,K106N, R107P, R107S, P108R, I110A, I110F, I110L, I110M, I110T, T111D,T111H, T111N, T111R, G112A, G112C, G112D, G112K, G112L, G112M, G112Q,G112R, G112S, G112T, G112Y, N127K, I131V, A132V, L133V, A136T, L137T,T138S, N140D, H142Q, Q14R, Y156H, K163T, D168E, H169R, Q175L, I183F,I183L, I183P, I183S, E184A, E184D, E184R, E184T, E184V, E185T, M187L,M189I, K191A, K191G, K191H, K191M, K191N, K191R, K191S, K191T, K191W,E197A, E197D, E197F, E197K, E197M, E197Q, E197S, E197T, E197V, I201C,I201E, I201F, I201H, I201L, I201S, I201T, I201V, H203K, L219M, L219W,F220L, V223I, F225Y, H230F, H230L, H230Y, N232S, Y246F, F249W, D250E,S274A, S274C, S274G, S274N, S274T, L278M, A280G, A280S, A280T, G281S,A282M, A282P, G284N, I285L, V303L, V303S, F306W, F306Y, S311N, K315E,D317E, D317K, I331C, I331L, I331N, I331S, I331T, I331V, N333T, P334N,P335T, L337T, L338A, L338Q, S341I, K373E, K373N, N375A, N375H, Y376C,Y376F, Y376L, K378G, K378P, K378Q, K378R, K380G, K380S, A382G, A382R,A382T, T383S, K384G, K384N, P386K, P386S, V387L, N389E, I405L, F407Y,S408D, S408N, N411R, D413S, D413V, Q416T, E419A, E419L, K420E, R421N,V424I, K427M, N429E, G432A and A436T. In some aspects, the amino acidsequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence of SEQ ID NO: 1, 2 or 3; or atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toone enzyme of Table 1. In certain aspects, the enzyme may comprise asubstitution at A282 (e.g., A282M or A282P), F306 (e.g., F306W or F306Y)or F249, such as F249W. In further aspects, the enzyme may comprise theL72N substitution. In still further aspects, the enzyme may comprise theF306W substitution. In specific aspects, the enzyme may comprise theH102W and N333T substitutions. In another aspect, the enzyme maycomprise a substitution at A99. In some aspects, the enzyme may comprisea substitution at G112. In certain specific aspects, the enzymecomprises a substitution at E103. In other aspects, the enzyme comprisesa substitution at V104. In further aspects, the enzyme may comprise asubstitution at S408. In several aspects, the enzyme may comprise theI183P substitution. In some particular aspects, the enzyme may comprisethe R107P substitution. In another specific aspect, the enzyme maycomprise the A436T substitution.

In still further aspects, the enzyme may comprise at least onesubstitution selected from L72N, H102W, A282P, F306W, I331S and N333T.In certain aspects, the enzyme comprises at least two, three, four orfive substitutions selected from L72N, H102W, A282P, F306W, I331S andN333T. In some aspects, the enzyme may comprise the substitutions L72N,H102W, A282P, F306W, I331S and N333T. In an additional aspect, theenzyme has a catalytic activity relative the KYN (kcat/kM) of at least8000M-1s-1. In other aspects, the enzyme has a catalytic activityrelative the KYN (kcat/kM) of between about 8000M-1s-1 and 40000 M-1s-1;10000M-1s-1 and 40000 M-1s-1; 20000M-1s-1 and 40000 M-1s-1; or25000M-1s-1 and 35000 M-1s-1.

In still further aspects, the polypeptide comprises an amino acidsequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence of one of the polypeptides ofTable 1. In specific aspects, the polypeptide comprises an amino acidsequence 100% identical to one of the polypeptides of Table 1. In yetstill further aspects, the polypeptide comprises an amino acid sequenceis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence of SEQ ID NO: 2. In specificaspects the polypeptide comprises an amino acid sequence 100% identicalto SEQ ID NO: 2. In certain aspects, the amino acid sequence is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to theamino acid sequence of SEQ ID NO: 3. In specific aspects the polypeptidecomprises an amino acid sequence 100% identical to SEQ ID NO: 3.

In a further aspect a kynureninase enzymes of the embodiments furthercomprises one of the amino acid substitutions disclosed inWO/2015/031771, WO/2016/033488 or WO/2017/151860, each of which isincorporated herein by reference in its entirety.

In further aspects, there are provided polypeptides comprising either anative or modified human or primate kynureninase capable of degradingKYN. In some embodiments, the polypeptides are capable of degrading KYNunder physiological conditions. For example, the polypeptides have acatalytic efficiency for KYN (k_(cat)/K_(M)) of at least or about 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000,6000, 7000, 8000, 9000, 10⁴, 10⁵, 10⁶ M⁻¹s⁻¹ or any range derivabletherein.

A modified polypeptide as discussed above may be characterized as havinga certain percentage of identity as compared to an unmodifiedpolypeptide (e.g., a native polypeptide, such as human kynureninase ofSEQ ID NO: 1) or to any polypeptide sequence disclosed herein. Forexample, the unmodified polypeptide may comprise at least, or up to,about 150, 200, 250, 300, 350, 400, 450 or 465 residues (or any rangederivable therein) of a native kynureninase. The percentage identity maybe about, at most or at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% (or any range derivable therein) between the modifiedand unmodified polypeptides, or between any two sequences in comparison.It is also contemplated that percentage of identity discussed above mayrelate to a particular modified region of a polypeptide as compared toan unmodified region of a polypeptide. For instance, a polypeptide maycontain a modified or mutant substrate recognition site of akynureninase that can be characterized based on the identity of theamino acid sequence of the modified or mutant substrate recognition siteof the kynureninase to that of an unmodified or mutant kynureninase fromthe same species or across the species. A modified or mutant humanpolypeptide characterized, for example, as having at least 90% identityto an unmodified kynureninase means that at least 90% of the amino acidsin that modified or mutant human polypeptide are identical to the aminoacids in the unmodified polypeptide.

Such an unmodified polypeptide may be a native kynureninase,particularly a human isoform or other primate isoforms. For example, thenative human kynureninase may have the sequence of SEQ ID NO: 1.Non-limiting examples of other native primate kynureninase include Pongoabelii kynureninase (Genbank ID: XP_009235962.1, GI: 686708656), Macacafascicularis kynureninase (Genbank ID: EHH54849.1, GI: 355750522), andPan troglodytes kynureninase (Genbank ID: XP_003309314.1, GI:332814521). Exemplary native polypeptides include a sequence havingabout, at most or at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identity (or any range derivable therein) of SEQ IDNO:1. For example, the native polypeptide may comprise at least or up toabout 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350,400, 450 or 465 residues (or any range derivable therein) of thesequence of SEQ ID NO:1.

In some aspects, polypeptides of the embodiments comprising akynureninase linked to a heterologous amino acid sequence. For example,the kynureninase may be linked to the heterologous amino acid sequenceas a fusion protein. In a particular embodiment, the kynureninase islinked to amino acid sequences, such as an IgG Fc, albumin, an albuminbinding protein, or an XTEN polypeptide for increasing the in vivohalf-life.

To increase serum persistence, the kynureninase may be linked to one ormore polyether molecules. In a particular embodiment, the polyether ispolyethylene glycol (PEG). The polypeptide may be linked (e.g.,covalently) to PEG via specific amino acid residues, such as lysine orcysteine. For therapeutic administration, such a polypeptide comprisingthe kynureninase may be dispersed in a pharmaceutically acceptablecarrier.

In some aspects, a nucleic acid encoding such a kynureninase iscontemplated. In some embodiments, the nucleic acid has been codonoptimized for expression in bacteria. In particular embodiments, thebacteria are E. coli. In other aspects, the nucleic acid has been codonoptimized for expression in fungus (e.g., yeast), insects, or mammals.In further aspects, there are provided vectors, such as expressionvectors, containing such nucleic acids. In particular embodiments, thenucleic acid encoding the kynureninase is operably linked to a promoter,including but not limited to heterologous promoters. In one embodiment,a kynureninase is delivered to a target cell by a vector (e.g., a genetherapy vector). Such viruses may have been modified by recombinant DNAtechnology to enable the expression of the kynureninase-encoding nucleicacid in the target cell. These vectors may be derived from vectors ofnon-viral (e.g., plasmids) or viral (e.g., adenovirus, adeno-associatedvirus, retrovirus, lentivirus, herpes virus, or vaccinia virus) origin.Non-viral vectors are preferably complexed with agents to facilitate theentry of the DNA across the cellular membrane. Examples of suchnon-viral vector complexes include the formulation with polycationicagents which facilitate the condensation of the DNA and lipid-baseddelivery systems. An example of a lipid-based delivery system wouldinclude liposome based delivery of nucleic acids.

In still further aspects, the present invention further contemplateshost cells comprising such vectors. The host cells may be bacteria(e.g., E. coli), fungal cells (e.g., yeast), insect cells, or mammaliancells.

In some embodiments, the vectors are introduced into host cells forexpressing the kynureninase. The proteins may be expressed in anysuitable manner. In one embodiment, the proteins are expressed in a hostcell such that the protein is glycosylated. In another embodiment, theproteins are expressed in a host cell such that the protein isaglycosylated.

In some embodiments, the cancer is any cancer that is sensitive tokynurenine depletion. In one embodiment, the present inventioncontemplates a method of treating a tumor cell or a cancer patientcomprising administering a formulation comprising such a polypeptide. Insome embodiments, the administration occurs under conditions such thatat least a portion of the cells of the cancer are killed. In anotherembodiment, the formulation comprises such a kynureninase withkynurenine-degrading activity at physiological conditions and furthercomprising an attached polyethylene glycol chain. In some embodiment,the formulation is a pharmaceutical formulation comprising any of theabove discussed kynureninases and pharmaceutically acceptableexcipients. Such pharmaceutically acceptable excipients are well knownto those of skill in the art. All of the above kynureninases may becontemplated as useful for human therapy.

In a further embodiment, there may also be provided a method of treatinga tumor cell comprising administering a formulation comprising anon-bacterial (mammalian, e.g., primate or mouse) kynureninase that haskynurenine-degrading activity or a nucleic acid encoding thereof.

In accordance with certain aspects of the present invention, such aformulation containing the kynureninase can be administeredintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intrasynovially, intratracheally, intranasally,intravitreally, intravaginally, intrarectally, intratumorally,intramuscularly, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularly, orally,topically, by inhalation, infusion, continuous infusion, localizedperfusion, via a catheter, via a lavage, in lipid compositions (e.g.,liposomes), or by other method or any combination of the forgoing aswould be known to one of ordinary skill in the art.

In a further embodiment, the method also comprises administering atleast a second anticancer therapy to the subject. The second anticancertherapy may be a surgical therapy, chemotherapy, radiation therapy,cryotherapy, hormone therapy, immunotherapy or cytokine therapy. Incertain aspects, the second anticancer therapy may be an immune checkpoint inhibitor therapy, such as anti-PD-1, anti-CTLA-4, anti-PD-L1antibody, anti-LAG3, anti-TIM-3, anti-ICOS, anti-CD137, anti-CD40,anti-KIR, anti-CD40L, anti-GITR, or anti-OX40 therapy. In furtheraspects, the second anticancer therapy may be an antibody therapy, suchwith an antibody-drug conjugate. In still further aspects, the secondanticancer therapy is an immunotherapy such as administering immuneeffector cells or an immunogenic composition. For example, theimmunogenic composition can comprise cancer cells antigens and,optionally, an adjuvant. In some aspects, immune effector cells cancomprise NK-cells, T-cells (e.g., CAR T-cells) or NK/T-cells.

In some embodiments, a T cell comprising a chimeric antigen receptor(CAR) and a kynureninase enzyme of the embodiments are contemplated foruse in treating a subject with cancer. In some aspects, the cell may betransfected with a DNA encoding the CAR and the kynureninase and, insome cases, a transposase.

The CAR may target any cancer-cell antigen of interest, including, forexample, HER2, CD19, CD20, and GD2. The antigen binding regions ordomain can comprise a fragment of the V_(H) and V_(L) chains of asingle-chain variable fragment (scFv) derived from a particular humanmonoclonal antibody, such as those described in U.S. Pat. No. 7,109,304,which is incorporated herein by reference in its entirety. The fragmentcan also be any number of different antigen binding domains of a humanantigen-specific antibody. In a more specific embodiment, the fragmentis an antigen-specific scFv encoded by a sequence that is optimized forhuman codon usage for expression in human cells. For additional examplesof CARs, see, for example, WO 2012/031744, WO 2012/079000, WO2013/059593, and U.S. Pat. No. 8,465,743, all of which are incorporatedherein by reference in their entireties.

The kynureninase may be any kynureninase disclosed herein. Methods oftransfecting of cells are well known in the art, but in certain aspects,highly efficient transfections methods such as electroporation areemployed. For example, nucleic acids may be introduced into cells usinga nucleofection apparatus. Preferably, the transfection step does notinvolve infecting or transducing the cells with virus, which can causegenotoxicity and/or lead to an immune response to cells containing viralsequences in a treated subject.

A wide range of CAR constructs and expression vectors for the same areknown in the art and are further detailed herein. For example, in someaspects, the CAR expression vector is a DNA expression vector such as aplasmid, linear expression vector or an episome. In some aspects, thevector comprises additional sequences, such as sequence that facilitatesexpression of the CAR, such a promoter, enhancer, poly-A signal, and/orone or more introns. In preferred aspects, the CAR coding sequence isflanked by transposon sequences, such that the presence of a transposaseallows the coding sequence to integrate into the genome of thetransfected cell.

In certain aspects, cells are further transfected with a transposasethat facilitates integration of a CAR coding sequence into the genome ofthe transfected cells. In some aspects, the transposase is provided asDNA expression vector. However, in preferred aspects, the transposase isprovided as an expressible RNA or a protein such that long-termexpression of the transposase does not occur in the transgenic cells.Any transposase system may be used in accordance with the embodiments.In other aspects, cells may be infected with a lentivirus to facilitateintegration of the CAR coding sequence and the kynureninase codingsequence into the genome of the cell.

In one embodiment, a composition comprising a kynureninase or a nucleicacid encoding a kynureninase is provided for use in the treatment of atumor in a subject. In another embodiment, the use of a kynureninase ora nucleic acid encoding a kynureninase in the manufacture of amedicament for the treatment of a tumor is provided. The kynureninasemay be any kynureninase of the embodiments.

Embodiments discussed in the context of methods and/or compositions ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

As used herein, “essentially free,” in terms of a specified component,is used herein to mean that none of the specified component has beenpurposefully formulated into a composition and/or is present only as acontaminant or in trace amounts. The total amount of the specifiedcomponent resulting from any unintended contamination of a compositionis preferably below 0.01%. Most preferred is a composition in which noamount of the specified component can be detected with standardanalytical methods.

As used herein in the specification and claims, “a” or “an” may mean oneor more. As used herein in the specification and claims, when used inconjunction with the word “comprising”, the words “a” or “an” may meanone or more than one. As used herein, in the specification and claim,“another” or “a further” may mean at least a second or more.

As used herein in the specification and claims, the term “about” is usedto indicate that a value includes the inherent variation of error forthe device, the method being employed to determine the value, or thevariation that exists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating certain embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-C: Successful outcome of HsKYN protein engineer. A) Humanvariants achieved >500 fold increase of catalytic activity(K_(cat)/K_(m)) for Kyn, comparable to bacterial tool Kynases. B) Humanvariants vastly improved catalytic stability in human serum in vitro. C)Human variants (68) and (153), as set forth in Table 1, herein, showedprolonged Kyn degradation in mice after a single iv dose.

FIGS. 2A-B: In vivo efficacy of HsKYN variant. A) Protein (68) incombination with antiPD-1 demonstrated significant tumor growthinhibition/survival benefits superior to Epacadostat, in the CT26 model.B) Complete responders (CRs) were re-challenged with CT26 and none ofthe CRs developed tumors, confirming anti-tumor memory against CT26. Incontrast, naive mice inoculated with CT26 developed tumors.

FIGS. 3A-B: Immunoprofiling of Protein (68) in CT26 tumor bearing mice.A) Multiplex gene expression analysis using Nanostring revealedupregulation of IFNγ gene signature upon HsKYN+/−antiPD1 treatment. B)FACs analysis demonstrated that HsKYN treatment increased CD8/Treg andM1/M2 macrophage ratios.

FIGS. 4A-C: The PD/PK profiles of Protein (153) in tox species. A)Protein (153) demonstrated robust and durable plasma Kyn depletion inrats after a single and repeated weekly iv dose(s) of 10 mg/kg. B)Protein (153) degraded plasma Kyn ≥50% for up to 7 days after a singleiv dose in cyno monkeys. C) Protein (153) monkey PK profile with afavorable T1/2>6 days.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. The Present Embodiments

In certain embodiments, the present disclosure provides kynureninases(Kynase), a Kyn depleting enzyme. In particular the application providesa set of human kynureninase variants that display at least 100-foldhigher catalytic activity towards Kynurenine degradation compared to thewild type human Kynureninase enzyme. In some aspects, kynureninasevariants provided herein additionally have increase resistance todeactivation in serum. Thus these enzymes have significantly improvedproperties that make them ideal for therapeutic disease intervention,such as the treatment of cancer. In some aspects, the enzymes (orsequences encoding the enzymes) can be used to treat cancer patients,such those with IDO1 and/or TDO2 expressing tumors.

In the present studies, the human Kynase (h-KYNase) was beensuccessfully engineered to exhibit vastly improved catalytic activityand stability toward Kyn over the WT enzyme, and achieved durable plasmaKyn depletion in mice. Single agent and PD1 combination efficacy wasalso demonstrated in CT26 and B16-IDO models, superior to the lead IDO1inhibitor Epacadostat, and the resulting complete responders (CRs) wereresistant to tumor rechallenge. Tumor immunoprofiling revealedupregulation of IFNg signature gene expressions and M1/M2 macrophageratio upon HsKYN+/−PD1 treatment. The lead HsKYN variant displayedfavorable Kyn degradation and PK profile in rats and non-human primates,and may be used for treatment of cancers, such as cancers where IDO1and/or TDO2 pathways play an immunosuppressive role.

II. Definitions

As used herein the terms “protein” and “polypeptide” refer to compoundscomprising amino acids joined via peptide bonds and are usedinterchangeably.

As used herein, the term “fusion protein” refers to a chimeric proteincontaining proteins or protein fragments operably linked in a non-nativeway.

As used herein, the term “half-life” (½-life) refers to the time thatwould be required for the concentration of a polypeptide thereof to fallby half in vitro or in vivo, for example, after injection in a mammal.

The terms “in operable combination,” “in operable order,” and “operablylinked” refer to a linkage wherein the components so described are in arelationship permitting them to function in their intended manner, forexample, a linkage of nucleic acid sequences in such a manner that anucleic acid molecule capable of directing the transcription of a givengene and/or the synthesis of desired protein molecule, or a linkage ofamino acid sequences in such a manner so that a fusion protein isproduced.

The term “linker” is meant to refer to a compound or moiety that acts asa molecular bridge to operably link two different molecules, wherein oneportion of the linker is operably linked to a first molecule, andwherein another portion of the linker is operably linked to a secondmolecule.

The term “PEGylated” refers to conjugation with polyethylene glycol(PEG), which has been widely used as a drug carrier, given its highdegree of biocompatibility and ease of modification. PEG can be coupled(e.g., covalently linked) to active agents through the hydroxy groups atthe end of the PEG chain via chemical methods; however, PEG itself islimited to at most two active agents per molecule. In a differentapproach, copolymers of PEG and amino acids have been explored as novelbiomaterial that would retain the biocompatibility of PEG, but thatwould have the added advantage of numerous attachment points permolecule (thus providing greater drug loading), and that can besynthetically designed to suit a variety of applications.

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor thereof. The polypeptide can be encoded by a full-lengthcoding sequence or by any portion of the coding sequence so as thedesired enzymatic activity is retained.

The term “native” refers to the typical form of a gene, a gene product,or a characteristic of that gene or gene product when isolated from anaturally occurring source. A native form is that which is mostfrequently observed in a natural population and is thus arbitrarilydesignated the normal or wild-type form. In contrast, the term“modified,” “variant,” or “mutant” refers to a gene or gene product thatdisplays modification in sequence and functional properties (i.e.,altered characteristics) when compared to the native gene or geneproduct.

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for an RNA capable of beingtranscribed. In some cases, RNA molecules are then translated into aprotein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

The term “therapeutically effective amount” as used herein refers to anamount of cells and/or therapeutic composition (such as a therapeuticpolynucleotide and/or therapeutic polypeptide) that is employed inmethods to achieve a therapeutic effect. The term “therapeutic benefit”or “therapeutically effective” as used throughout this applicationrefers to anything that promotes or enhances the well-being of thesubject with respect to the medical treatment of this condition. Thisincludes, but is not limited to, a reduction in the frequency orseverity of the signs or symptoms of a disease. For example, treatmentof cancer may involve, for example, a reduction in the size of a tumor,a reduction in the invasiveness of a tumor, reduction in the growth rateof the cancer, or prevention of metastasis. Treatment of cancer may alsorefer to prolonging survival of a subject with cancer.

The term “K_(M)” as used herein refers to the Michaelis-Menten constantfor an enzyme and is defined as the concentration of the specificsubstrate at which a given enzyme yields one-half its maximum velocityin an enzyme catalyzed reaction. The term “k_(cat)” as used hereinrefers to the turnover number or the number of substrate molecules eachenzyme site converts to product per unit time, and in which the enzymeis working at maximum efficiency. The term “k_(cat)/K_(M)” as usedherein is the specificity constant, which is a measure of howefficiently an enzyme converts a substrate into product.

The term “chimeric antigen receptors (CARs),” as used herein, may referto artificial T-cell receptors, chimeric T-cell receptors, or chimericimmunoreceptors, for example, and encompass engineered receptors thatgraft an artificial specificity onto a particular immune effector cell.CARs may be employed to impart the specificity of a monoclonal antibodyonto a T cell, thereby allowing a large number of specific T cells to begenerated, for example, for use in adoptive cell therapy. In specificembodiments, CARs direct specificity of the cell to a tumor associatedantigen, for example. In some embodiments, CARs comprise anintracellular activation domain, a transmembrane domain, and anextracellular domain comprising a tumor associated antigen bindingregion. In particular aspects, CARs comprise fusions of single-chainvariable fragments (scFv) derived from monoclonal antibodies (such asthose described in U.S. Pat. No. 7,109,304, which is incorporated hereinby reference in its entirety), fused to CD3-zeta transmembrane andendodomains. The specificity of other CAR designs may be derived fromligands of receptors (e.g., peptides) or from pattern-recognitionreceptors, such as Dectins. In particular embodiments, one can targetmalignant B cells by redirecting the specificity of T cells by using aCAR specific for the B-lineage molecule, CD19. In certain cases, thespacing of the antigen-recognition domain can be modified to reduceactivation-induced cell death. In certain cases, CARs comprise domainsfor additional co-stimulatory signaling, such as CD3-zeta, FcR, CD27,CD28, CD137, DAP10, and/or OX40. In some cases, molecules can beco-expressed with the CAR, including co-stimulatory molecules, reportergenes for imaging (e.g., for positron emission tomography), geneproducts that conditionally ablate the T cells upon addition of apro-drug, homing receptors, chemokines, chemokine receptors, cytokines,and cytokine receptors.

“Treatment” and “treating” refer to administration or application of atherapeutic agent to a subject or performance of a procedure or modalityon a subject for the purpose of obtaining a therapeutic benefit of adisease or health-related condition. For example, a treatment mayinclude administration of a pharmaceutically effective amount of akynureninase.

“Subject” and “patient” refer to either a human or non-human, such asprimates, mammals, and vertebrates. In particular embodiments, thesubject is a human.

III. Kynureninase Polypeptides

Some embodiments concern modified proteins and polypeptides. Particularembodiments concern a modified protein or polypeptide that exhibits atleast one functional activity that is comparable to the unmodifiedversion, preferably, the kynurenine degrading activity. In furtheraspects, the protein or polypeptide may be further modified to increaseserum stability. Thus, when the present application refers to thefunction or activity of “modified protein” or a “modified polypeptide,”one of ordinary skill in the art would understand that this includes,for example, a protein or polypeptide that possesses an additionaladvantage over the unmodified protein or polypeptide, such as kynureninedegrading activity or thermodynamic stability.

Determination of activity may be achieved using assays familiar to thoseof skill in the art, particularly with respect to the protein'sactivity, and may include for comparison purposes, the use of nativeand/or recombinant versions of either the modified or unmodified proteinor polypeptide.

In certain embodiments, a modified polypeptide, such as a modifiedkynureninase, may be identified based on its increase in kynurenine. Forexample, substrate recognition sites of the unmodified polypeptide maybe identified. This identification may be based on structural analysisor homology analysis. A population of mutants involving modifications ofsuch substrate recognition sites may be generated. In a furtherembodiment, mutants with increased kynurenine degrading activity may beselected from the mutant population. Selection of desired mutants mayinclude methods, such as detection of byproducts or products fromkynurenine degradation.

Modified proteins may possess deletions and/or substitutions of aminoacids; thus, a protein with a deletion, a protein with a substitution,and a protein with a deletion and a substitution are modified proteins.In some embodiments, these modified proteins may further includeinsertions or added amino acids, such as with fusion proteins orproteins with linkers, for example. A “modified deleted protein” lacksone or more residues of the native protein, but may possess thespecificity and/or activity of the native protein. A “modified deletedprotein” may also have reduced immunogenicity or antigenicity. Anexample of a modified deleted protein is one that has an amino acidresidue deleted from at least one antigenic region that is, a region ofthe protein determined to be antigenic in a particular organism, such asthe type of organism that may be administered the modified protein.

Substitution or replacement variants typically contain the exchange ofone amino acid for another at one or more sites within the protein andmay be designed to modulate one or more properties of the polypeptide,particularly its effector functions and/or bioavailability.Substitutions may or may not be conservative, that is, one amino acid isreplaced with one of similar shape and charge. Conservativesubstitutions are well known in the art and include, for example, thechanges of: alanine to serine; arginine to lysine; asparagine toglutamine or histidine; aspartate to glutamate; cysteine to serine;glutamine to asparagine; glutamate to aspartate; glycine to proline;histidine to asparagine or glutamine; isoleucine to leucine or valine;leucine to valine or isoleucine; lysine to arginine; methionine toleucine or isoleucine; phenylalanine to tyrosine, leucine, ormethionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

In addition to a deletion or substitution, a modified protein maypossess an insertion of residues, which typically involves the additionof at least one residue in the polypeptide. This may include theinsertion of a targeting peptide or polypeptide or simply a singleresidue. Terminal additions, called fusion proteins, are discussedbelow.

The term “biologically functional equivalent” is well understood in theart and is further defined in detail herein. Accordingly, sequences thathave between about 70% and about 80%, or between about 81% and about90%, or even between about 91% and about 99% of amino acids that areidentical or functionally equivalent to the amino acids of a controlpolypeptide are included, provided the biological activity of theprotein is maintained. A modified protein may be biologicallyfunctionally equivalent to its native counterpart in certain aspects.

It also will be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

In certain embodiments, a kynureninase according to the embodimentscomprises and amino acid sequence at least about 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the kynureninase of SEQ ID NOs: 1, 2 or 3. In still further aspects,a kynureninase is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the kynureninaseof SEQ ID NOs: 1, 2 or 3 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 of theamino acid substitutions selected from the group consisting of LBP,K38E, Y47L, I48F, K50Q, I51M, S60N, K64N, D65G, E66K, N67D, N67P, D67S,A68F, A68T, A68V, F71L, F71M, L72N, K84E, E88N, E89K, E89S, E90Q, D92E,K93N, K93T, A95H, A95Q, K96N, I97H, I97L, I97V, A98G, A99G, A99I, A99R,A99S, A99T, A99V, Y100N, Y100S, Y100T, G101A, H102W, E103F, E103H,E103N, E103Q, E103R, E103V, E103W, V104D, V104E, V104F, V104H, V104K,V104L, V104R, G105A, G105H, G105S, G105T, E106D, K106D, K106E, K106H,K106N, R107P, R107S, P108R, I110A, I110F, I110L, I110M, I110T, T111D,T111H, T111N, T111R, G112A, G112C, G112D, G112K, G112L, G112M, G112Q,G112R, G112S, G112T, G112Y, N127K, I131V, A132V, L133V, A136T, L137T,T138S, N140D, H142Q, Q14R, Y156H, K163T, D168E, H169R, Q175L, I183F,I183L, I183P, I183S, E184A, E184D, E184R, E184T, E184V, E185T, M187L,M189I, K191A, K191G, K191H, K191M, K191N, K191R, K191S, K191T, K191W,E197A, E197D, E197F, E197K, E197M, E197Q, E197S, E197T, E197V, I201C,I201E, I201F, I201H, I201L, I201S, I201T, I201V, H203K, L219M, L219W,F220L, V223I, F225Y, H230F, H230L, H230Y, N232S, Y246F, F249W, D250E,S274A, S274C, S274G, S274N, S274T, L278M, A280G, A280S, A280T, G281S,A282M, A282P, G284N, I285L, V303L, V303S, F306W, F306Y, S311N, K315E,D317E, D317K, I331C, I331L, I331N, I331S, I331T, I331V, N333T, P334N,P335T, L337T, L338A, L338Q, S341I, K373E, K373N, N375A, N375H, Y376C,Y376F, Y376L, K378G, K378P, K378Q, K378R, K380G, K380S, A382G, A382R,A382T, T383S, K384G, K384N, P386K, P386S, V387L, N389E, I405L, F407Y,S408D, S408N, N411R, D413S, D413V, Q416T, E419A, E419L, K420E, R421N,V424I, K427M, N429E, G432A and A4361. In further aspects, a kynureninaseis at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical to one of the kynureninases providedin Table 1. In particular aspects, the kynureninase is selected from oneof those provided in Table 1. In some aspects, a kynureninase, e.g.,according to Table 1, may or may not be PEGylated.

Table 1, below, sets forth various kynureninase enzymes that may be usedin conjunction with the compositions and methods of the presentdisclosure. Although not listed explicitly in Table 1, “Protein (1)”herein refers to wild-type human kynureninase, the amino acid sequenceof which is set forth in SEQ ID NO: 1.

TABLE 1 Exemplary kynureninase enzymes of the embodiments. Number ofProtein Mutations List of Mutations Tag PEGylated 2 9N67D/L72N/E103Q/M189I/F225Y/S274G/I331V/I405L/S408N His NO 3 9N67D/L72N/E103Q/M189I/F225Y/S274G/I331V/I405L/S408N His YES 4 7A99I/G112A/F306Y/I331N/I405L/S408N/A436T His NO 5 7A99I/G112A/F306Y/I331N/I405L/S408N/A436T His YES 6 10Q14R/A99I/G112A/M189I/H230Y/F306Y/I331N/I405L/S408N/A436T His NO 7 10Q14R/A99I/G112A/M189I/H230Y/F306Y/I331N/I405L/S408N/A436T His YES 8 17N67D/L72N/E103Q/A99I/G112A/M189I/F225Y/S274G/I331V/F306Y/K380S/ His NOA382R/K384N/P386K/I405L/S408N/A436T 9 17N67D/L72N/E103Q/A99I/G112A/M189I/F225Y/S274G/I331V/F306Y/K380S/ His YESA382R/K384N/P386K/I405L/S408N/A436T 10 18N67D/L72N/A99I/H102W/E103F/V104E/K106E/R107S/I110M/A136T/M189I/ His NOF225Y/S274G/I331C/N333T/S341I/I405L/S408N 11 27N67D/L72N/A99I/H102W/E103F/V104E/K106E/R107S/I110M/I131V/L133V/ His NOA136T/T138S/M189I/F225Y/G274T/L278M/G281S/A282P/F306W/K315E/D317E/I331C/N333T/S341I/I405L/S408N 12 25N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ His NOA132V/A136T/M189I/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/I405L/S408N 13 25N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ His YESA132V/A136T/M189I/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/I405L/S408N 14 8A99I/G112A/G284N/I285L/F306Y/I331N/I405L/S408N/A436T His NO 15 7A99I/G112A/K191N/F306Y/I331N/I405L/S408N/A436T His NO 16 10L8P/A99I/G112A/Q175L/F306Y/I331N/K373E/K378R/I405L/S408N/A436T His NO 1710 A99I/G112A/F306Y/I331N/K380S/A382R/K384N/P386K/I405L/S408N/A436T HisNO 18 9 A99I/G112A/F306Y/I331N/N375H/Y376L/K378P/I405L/S408N/A436T HisNO 19 22 N67D/A68T/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110L/T111H/His NO G112Y/A136T/M189I/F225Y/S274G/F306W/I331C/N333T/S341I/I405L/S408N20 23 N67D/L72N/A99I/H102W/E103F/V104E/K106E/R107S/I110M/A132V/A136T/His NO M189I/F225Y/G274S/A280S/G281S/A282P/F306W/I331C/N333T/S341I/I405L/S408N 21 23N67D/F71L/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/G112Y/A136T/ HisNO M189I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/I405L/S408N 22***sequencing was not fully resolved*** His NO 23 26D67N/A68V/L72N/A99I/H102W/E103F/V104E/K106E/R107S/M110I/T111H/G112Y/ HisNO A136T/M189I/F225Y/G274T/A280S/G281S/A282P/F306W/K315E/I331C/N333T/S341I/I405L/S408N 24 25D67N/L72N/A99I/H102W/E103F/V104E/K106E/R107S/I110M/T111H/G112Y/ His NOA136T/M189I/F225Y/G274S/A280S/G281S/A282P/F306W/K315E/I331C/N333T/S341I/I405L/S408N 25 29N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ His NOA132V/A136T/M189I/V223I/F225Y/S274G/A280S/G281S/A282P/V303L/F306W/S311N/K315E/I331C/N333T/S341I/I405L/S408N 26 23D67S/L72N/A99I/H102W/E103F/V104E/K106E/R107S/I110M/A132V/L133V/ His NOA136T/M189I/F225Y/G27A4/A280S/A282P/F306W/I331C/N333T/S341I/ I405L/S408N27 24 D67N/L72N/A99I/H102W/E103F/V104E/K106E/R107S/I110M/T111H/G112Y/His NO A136T/M189I/F225Y/G274N/L278M/A282P/F306W/K315E/I331C/N333T/S341I/I405L/S408N 28 22D67N/A68V/L72N/A99I/H102W/E103F/V104E/K106E/R107S/M110I/A136T/ His NOM189I/F225Y/G274T/G281S/A282P/F306W/I331C/N333T/S341I/I405L/S408N 29 27N67D/F71L/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ HisNO A136T/M189I/V223I/F225Y/G274S/A280S/G281S/A282P/F306W/K315E/I331C/N333T/S341I/I405L/S408N 30 25D67N/L72N/A99I/H102W/E103F/V104E/K106E/R107S/I110M/T111H/G112Y/ His NOA136T/M189I/F225Y/G274S/A280S/G281S/A282P/F306W/D317E/I331C/N333T/S341I/I405L/S408N 31 29N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ His NOA132V/A136T/V223I/M189I/F225Y/S274G/A280S/G281S/A282P/V303S/F306W/K315E/D317K/I331C/N333T/S341I/I405L/S408N 32 24N67D/A68T/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ HisNO A136T/M189I/F225Y/G274N/A280S/A282P/F306W/I331C/N333T/S341I/I405L/S408N 33 24N67D/A68T/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ HisYES A136T/M189I/F225Y/G274N/A280S/A282P/F306W/I331C/N333T/S341I/I405L/S408N 34 20L72N/A99V/Y100N/H102W/E103F/V104E/K106D/R107S/T111H/G112Y/L133V/ His NOA136T/F225Y/S274N/A280S/G281S/A282P/F306W/I331C/N333T 35 21L72N/A99V/Y100N/H102W/E103F/V104E/K106D/R107S/T111N/G112Y/I131V/ His NOL133V/A136T/F225Y/S274T/A280S/G281S/A282P/F306W/I331C/N333T 36 22L72N/A99V/Y100N/H102W/E103F/V104E/K106D/R107S/I110M/T111N/G112Y/ His NOI131V/L133V/A136T/F225Y/S274N/A280S/G281S/A282P/F306W/I331C/N333T 37 22N67D/F71L/L72N/A99V/Y100N/H102W/E103F/V104E/K106D/R107S/I110M/ His NOT111H/G112Y/A136T/F225Y/S274G/A280S/G281S/A282P/F306W/I331C/N333T 38 21L72N/E90Q/A99V/Y100N/H102W/E103F/V104E/K106D/R107S/I110M/T111D/ His NOG112Y/A136T/F225Y/S274N/A280S/G281S/A282M/F306W/I331C/N333T 39 16Y100T/H102W/E103F/V104E/K106D/R107S/T111H/G112Y/A136T/M189I/F225Y/ HisNO A280G/A282P/F306W/I331C/N333T 40 19A99S/Y100S/G101A/H102W/E103F/V104E/K106D/R107S/I110M/T111H/G112Y/ His NOA136T/F225Y/A280T/A282P/F306W/I331C/N333T/K373N 41 3 K191S-E197T-I201HHis NO 42 3 K191R-E197S-I201T His NO 43 3 K191S-E197T-I201T His NO 44 2I183P-E184A His NO 45 3 K191R-E197F-I201T His NO 46 2 K191R-I201E His NO47 3 K191W-E197V-I201E His NO 48 3 K191H-E197V-I201E His NO 49 2E197M-I201L His NO 50 3 K191G-E197V-I201H His NO 51 3 K191T-E197Q-I201EHis NO 52 3 K191H-E197V-I201E His NO 53 25N67D/A68T/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ HisNO A136T/M189I/F225Y/G274N/A280S/A282P/F306W/I331C/N333T/S341I/K373N/I405L/S408N 54 22L72N/E90Q/A99V/Y100N/H102W/E103F/V104E/K106D/R107S/I110M/T111D/ His NOG112Y/A136T/F225Y/S274N/A280S/G281S/A282M/F306W/I331C/N333T/K373N 55 37N67D/A68T/L72N/E88N/E89K/D92E/K93T/A95Q/K96N/I97H/A98G/A99I/H102W/ HisNO E103F/V104E/E106D/R107S/M110L/T111H/G112Y/A136T/M189I/F225Y/A280S/A282P/F306W/I331L/N333T/P335T/L338A/S341I/I405L/S408N/E419A/K420E/R421N/V424I 56 36 (counts Y47L/I48F/K50Q/del51- His NO deletion of62/K64N/D65G/E66K/N67P/A68F/L72N/E88N/E89S/E90Q/K93N/K96N/I97L/A99I/ 10residuesH102W/E103F/V104E/E106D/R107S/M110L/T111H/G112Y/A136T/M189I/F225Y/ asone A280G/A282P/F306W/I331C/N333T/S341I/I405L/S408N mutation) 57 29Q14R/K38E/I51M/N67D/A68T/L72N/A99I/H102W/E103F/V104E/E106D/R107S/ His NOM110L/T111H/G112Y/N127K/A136T/M189I/K191N/E197T/F225Y/H230F/S274G/F306W/I331C/N333T/S341I/I405L/S408N 58 29S60N/N67D/L72N/K84E/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/ HisNO G112Y/A132V/A136T/Y156H/K163T/M189I/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/I405L/S408N 59 26N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ His NOA132V/A136T/M189I/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/L338Q/S341I/I405L/S408N 60 26N67D/L72N/A99I/Y100N/H102W/E103F/V104E/E106D/R107S/M110I/T111H/ His NOG112Y/A132V/A136T/M189I/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/I405L/S408N 61 27N67D/L72N/I97V/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ HisNO A132V/A136T/N140D/M189I/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/I405L/S408N 62 28N67D/A68T/L72N/E90Q/K93N/A95H/K96N/I97L/A99I/H102W/E103F/V104E/ His NOE106D/R107S/M110L/T111H/G112Y/A136T/M189I/F225Y/A280S/A282P/F306W/I331C/N333T/S341I/I405L/S408N 63 25N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ His NOA132V/A136T/M189I/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/I405L/S408N 64 26N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ His NOA132V/A136T/D168E/M189I/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/I405L/S408N 65 26N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ His NOA132V/A136T/M189I/V223I/F225Y/D250E/S274G/A280S/G281S/A282P/I331C/N333T/S341I/I405L/S408N 66 25N67D/A68T/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/ His NOG112Y/A136T/D168E/M189I/F225Y/G274N/A280S/A282P/F306W/I331C/N333T/S341I/I405L/S408N 67 25N67D/A68T/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/ His NOG112Y/A136T/M189I/F225Y/D250E/G274N/A280S/A282P/F306W/I331C/N333T/S341I/I405L/S408N 68 8L72N/A99I/H102W/G112Y/A282P/F306W/I331S/N333T His NO 69 8L72N/A99I/H102W/G112Y/A282P/F306W/I331S/N333T His YES 70 9L72N/A99I/H102W/G112Y/A282P/F306W/I331S/N333T/P334N His NO 71 16L72N/A99I/H102W/E103F/E106D/M110I/T111H/G112Y/A132V/A136T/M189I/ His NOA282P/I331C/N333T/S341I/S408N 72 19N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/T111H/G112Y/A136T/ His NOM189I/A280S/A282P/F306W/I331C/N333T/I405L/S408N 73 28N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ His NOA132V/A136T/M189I/K191S/E197T/I201H/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/I405L/S408N 74 28N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ His NOA132V/A136T/M189I/K191R/E197F/I201T/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/I405L/S408N 75 28N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ His NOA132V/A136T/M189I/K191SW/E197V/I201E/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/I405L/S408N 76 27N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ His NOA132V/A136T/M189I/E197M/I201L/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/I405L/S408N 77 28N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ His NOA132V/A136T/M189I/K191H/E197V/I201E/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/I405L/S408N 78 28N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ His NOA132V/A136T/M189I/K191R/E197S/I201T/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/I405L/S408N 79 28N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ His NOA132V/A136T/M189I/K191G/E197V/I201H/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/I405L/S408N 80 28N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ His NOA132V/A136T/M189I/K191S/E197T/I201T/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/I405L/S408N 81 28N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ His NOA132V/A136T/M189I/K191G/E197A/I201E/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/I405L/S408N 82 12L72N/A99S/H102W/E103F/V104K/K106H/R107S/G112Y/A282P/F306W/I331C/ His NON333T 83 12N67D/L72N/A99I/H102W/K106D/S274G/A280S/G281S/A282P/F306W/I331S/ His NON333T 84 24N67D/A68T/L72N/A99I/H102W/E103F/V104E/K106D/R107S/I110L/T111H/ His NOG112Y/A136T/M189I/F225Y/S274N/A280S/A282P/F306W/I331C/N333T/S341I/I405L/S408N 85 13L72N/A99I/H102W/E103V/V104E/K106D/R107S/T111H/G112Y/A282P/F306W/ His NOI331S/N333T 86 12L72N/A99V/H102W/E103F/V104E/K106N/T111N/A280S/A282P/F306W/I331S/ His NON333T 87 10 L72N/A99I/H102W/G112Y/A282P/F306W/I331S/N333T/T383S/D413VHis NO 88 13L72N/A99I/Y100N/H102W/G112Y/H169R/M187L/Y246F/A282P/F306W/I331S/ His NON333T/A382T 89 11L72N/A99I/H102W/V104R/G105H/R107P/G112Y/A282P/F306W/I331S/N333T His NO90 11 L72N/H102W/E103N/K106D/R107P/T111R/G112Y/A282P/F306W/I331T/N333THis NO 91 10 L72N/A99I/H102W/G112Y/A282P/F306W/I331S/N333T/Y376F/K378QHis NO 92 11L72N/A99I/H102W/G112Y/K191S/E197D/I201E/A282P/F306W/I331S/N333T His NO93 10 L72N/A99I/H102W/G112Y/K191A/I201V/A282P/F306W/I331S/N333T His NO94 15 L72N/A99I/H102W/G112Y/I183P/E184A/M187L/M189I/K191N/E197T/I201T/His NO A282P/F306W/I331S/N333T 95 His NO 96 15F71M/L72N/E103F/V104H/G105T/I183P/E184A/M187L/M189I/K191N/E197T/ His NOI201T/A280S/A282P/F306Y 97 15F71M/L72N/E103F/V104H/G105T/I183P/E184A/M187L/M189I/K191N/E197T/ His YESI201T/A280S/A282P/F306Y 98 7 L72N/H102W/G112Y/A282P/F306W/I331S/N333THis NO 99 9 L72N/G101A/H102W/G112Y/E184A/A282P/F306W/I331S/N333T His NO100 10 L72N/A99I/H102W/G112Y/I183P/E184A/A282P/F306W/I331S/N333T His NO101 12 L72N/A99I/H102W/G112Y/I183L/M189I/K191S/I201T/A282P/F306W/I331S/His NO N333T 102 12L72N/A99I/H102W/G112Y/M187L/M189I/K191R/I201T/A282P/F306W/I331S/ His NON333T 103 13L72N/A99V/E103Q/H102W/G112Y/I183P/E184A/E197A/I201T/A282P/F306W/ His NOI331S/N333T 104 13L72N/A99I/H102W/E103Q/V104D/G105S/G112Y/I183P/E184A/A282P/F306W/ His NOI331S/N333T 105 9 L72N/A99I/H102W/G112Y/I183P/A282P/F306W/I331S/N333THis NO 106 13L72N/A99V/H102W/E103Q/V104H/G112Y/I183P/M189I/K191S/A282P/F306W/ His NOI331S/N333T 107 11L72N/H102W/R107P/G112Y/I183P/E184A/E197A/A282P/F306W/I331S/N333T His NO108 11 L72N/A99T/H102W/G112Y/I183P/E184A/I201T/A282P/F306W/I331S/N333THis NO 109 12L72N/A99I/H102W/G112Y/A136T/L137T/F225Y/A282P/F306W/I331S/N333T/ His NOF407Y 110 10L72N/A99I/H102W/G112Y/A282P/F306W/I331S/N333T/I405L/S408D/A436T His NO111 13 L72N/A99I/H102W/G112Y/I183P/E184A/M187L/M189I/K191N/A282P/F306W/His NO I331S/N333T 112 13L72N/A99V/H102W/E103Q/G112Y/I183P/E184A/E197A/I201T/A282P/F306W/ His NOI331S/N333T 113 15L72N/A99V/H102W/E103Q/V104H/G105A/R107P/G112Y/M187L/K191S/I201T/ His NOA282P/F306W/I331S/N333T 114 15L72N/A99V/H102W/E103Q/V104H/G105A/R107P/G112Y/M187L/K191S/I201T/ His YESA282P/F306W/I331S/N333T 115 14L72N/H102W/V104D/G105A/G112Y/I183F/E184T/M189I/K191S/I201T/A282P/ His NOF306W/I331S/N333T 116 14L72N/A99V/H102W/V104H/G105A/G112Y/M187L/M189I/K191S/I201T/A282P/ His NOF306W/I331S/N333T 117 14L72N/A99I/H102W/V104D/G105S/G112Y/I183P/M187L/M189I/I201T/A282P/ His NOF306W/I331S/N333T 118 16L72N/A99V/H102W/E103Q/V104D/G112Y/I183P/E184A/M189I/K191R/I201T/ His NOA280S/A282P/F306W/I331S/N333T 119 12L72N/A99I/H102W/E103Q/V104H/G112Y/I183L/M189I/A282P/F306W/I331S/ His NON333T 120 13L72N/A99I/H102W/G112Y/I183S/E183A/M189I/K191N/I201T/A282P/F306W/ His NOI331S/N333T 121 10L72N/A99I/H102W/G112Y/A136T/L137T/A282P/F306W/I331S/N333T His NO 122 13L72N/A99I/H102W/G112Y/A136T/L137T/F220L/F225Y/H230L/A282P/F306W/ His NOI331S/N333T 123 11L72N/A99I/H102W/G112Y/L137T/F220L/H203K/A282P/F306W/I331S/N333T His NO124 13 L72N/A99I/H102W/G112Y/A136T/L137T/F225Y/A282P/F306W/I331S/N333T/His NO F407Y 125 11L72N/A99I/H102W/R107P/G112Y/I183L/E184A/A282P/F306W/I331S/N333T His NO126 15 L72N/A99V/H102W/E103Q/V104H/G105S/R107P/G112Y/M187L/K191R/I201T/His NO A282P/F306W/I331S/N333T 127 15L72N/A99I/H102W/G112Y/I183L/E184R/M187L/M189I/K191N/E197K/I201T/ His NOA282P/F306W/I331S/N333T 128 13L72N/A99I/H102W/G112Y/E184D/M187L/M189I/K191R/E197A/I201T/A282P/ His NOF306W/I331S/N333T 129 13L72N/A99I/H102W/G112Y/I183S/E184V/M187L/M189I/K191R/A282P/F306W/ His NOI331S/N333T 130 14L72N/A99V/H102W/V104D/G112Y/E184A/M187L/M189I/K191R/E197A/A282P/ His NOF306W/I331S/N333T 131 15L72N/A99V/H102W/V104H/G105A/G112Y/M189I/K191S/I201T/A282P/F306W/ His NOI331S/N333T 132 16L72N/A99I/H102W/E103Q/V104D/G105S/G112Y/I183P/M189I/K191S/I201T/ His NOA280S/A282P/F306W/I331S/N333T 133 13L72N/A99V/H102W/E103Q/R107P/G112Y/I183P/E184A/I201T/A282P/F306W/ His NOI331S/N333T 134 14L72N/A99T/H102W/E103F/R107P/G112Y/I183P/E184A/I201T/A282P/S724C/ His NOF306W/I331S/N333T 135 9L72N/A99V/H102W/R107P/G112Y/A282P/F306W/I331S/N333T His NO 136 17L72N/A99V/H102W/E103Q/V104H/G105A/R107P/G112Y/M187L/K191S/I201T/ His NOA282P/F306W/I331S/N333T/I405L/S408D 137 16L72N/A99V/H102W/E103Q/V104D/G112Q/I183P/E184A/M189I/K191R/I201T/ His NOA280S/A282P/F306W/I331S/N333T 138 14L72N/A99V/H102W/E103Q/V104H/R107P/G112Y/I183P/A282P/F306W/I331S/ His NON333T/S408N/A436T 139 17L72N/A99V/H102W/E103Q/V104H/G105A/R107P/G112L/M187L/K191S/I201T/ His NOF220L/F225Y/A282P/F306W/I331S/N333T 140 18L72N/A99V/H102W/E103Q/V104H/G105A/R107P/G112L/A136T/M187L/K191S/ His NOI201T/H230L/A282P/F306W/I331S/N333T 141 18L72N/A99V/H102W/E103Q/V104H/G105A/R107P/G112C/M187L/K191S/I201T/ His NOF220L/F225Y/A282P/F306W/I331S/N333T 142 16L72N/A99V/H102W/E103Q/V104D/G112R/I183P/E184A/M189I/K191R/I201T/ His NOA280S/A282P/F306W/I331S/N333T 143 12L72N/A99V/H102W/V104H/G105A/M189I/K191S/I201T/A282P/F306W/I331S/ His NON333T 144 12L72N/A99V/H102W/E103Q/V104D/R107P/G112C/I201E/A282P/F306W/I331S/ His NON333T 145 12L72N/A99V/H102W/E103Q/V104D/G112L/M187L/I201T/A282P/F306W/I331S/ His NON333T 146 12L72N/A99V/H102W/E103Q/V104L/G112D/K191S/A282P/F306W/I331S/N333T/ His NOS408N 147 14L72N/A99I/H102W/E103Q/V104L/G112Y/A136T/K191S/A282P/F306W/I331S/ His NON333T/S408N/A436T 148 15L72N/A99V/H102W/E103Q/V104L/R107P/G112Y/A136T/I183P/K191S/I201T/ His NOA282P/F306W/I331S/N333T 149 14L72N/A99V/H102W/E103Q/V104H/R107P/G112Y/H142Q/K191S/I201T/A282P/ His NOF306W/I331S/N333T 150 14L72N/A99I/H102W/E103Q/V104H/G112D/K191S/I201T/F225Y/A282P/F306W/ His NOI331S/N333T/S408N 151 15L72N/A99V/H102W/E103Q/V104H/G105A/R107P/G112Y/M187L/K191S/I201T/ His NOA282P/F306W/I331S/N333T/S408D 152 14L72N/A99G/H102W/E103H/V104H/R107P/G112Y/I183P/A282P/F306W/I331S/ His NON333T/S408N/A436T 153 14L72N/A99R/H102W/E103R/V104H/R107P/G112Y/I183P/A282P/F306W/I331S/ His NON333T/S408N/A436T 154 14L72N/A99G/H102W/E103W/V104H/R107P/G112Y/I183P/A282P/F306W/I331S/ His NON333T/S408N/A436T 155 13L72N/A99V/H102W/E103Q/V104H/R107P/I183P/A282P/F306W/I331S/N333T/ His NOS408N/A436T 156 15L72N/A99V/H102W/E103Q/V104H/R107P/G112Y/N140D/I183P/A282P/F306W/ His NOI331S/N333T/S408N/A436T 157 15L72N/A99V/H102W/E103Q/V104H/R107P/I110F/G112Y/I183P/A282P/F306W/ His NOI331S/N333T/S408N/A436T 158 14L72N/A99V/H102W/E103Q/V104H/R107P/G112L/I183P/A282P/F306W/I331S/ His NON333T/S408N/A436T 159 14L72N/A99V/H102W/E103Q/V104H/R107P/G112K/I183P/A282P/F306W/I331S/ His NON333T/S408N/A436T 160 15L72N/A99V/H102W/E103Q/V104H/R107P/T111R/G112R/I183P/A282P/F306W/ His NOI331S/N333T/S408N/A436T 161 15L72N/A99V/H102W/E103Q/V104H/R107P/G112Y/I183P/L219M/A282P/F306W/ His NOI331S/N333T/S408N/A436T 162 16L72N/A99V/H102W/E103Q/V104H/G105A/R107P/T111N/Y112A/I183P/A282P/ His NOF306W/I331S/N333T/S408N/A436T 163 19L72N/A99V/H102W/E103Q/V104H/R107P/G112Y/I183P/E184A/M189I/K191R/ His NOI201T/L219W/A282P/F306W/I331S/N333T/S408N/A436T 164 15L72N/A99V/H102W/E103Q/V104H/R107P/G112Y/D168E/I183P/A282P/F306W/ His NOI331S/N333T/S408N/A436T 165 19L72N/A99V/H102W/E103Q/V104H/R107P/G112Y/I183P/E184A/M189I/K191R/ His NOI201T/F249W/A282P/F306W/I331S/N333T/S408N/A436T 166 15L72N/A99V/H102W/E103Q/V104H/R107P/G112Y/I183P/F249W/A282P/F306W/ His NOI331S/N333T/S408N/A436T 167 14 Identical AA sequence to (138) His NO 16815 L72N/A99R/H102W/E103R/V104H/R107P/G112Y/I183P/F249W/A282P/F306W/ HisNO I331S/N333T/S408N/A436T 169 15L72N/A99G/H102W/E103W/V104H/R107P/G112Y/I183P/F249W/A282P/F306W/ His NOI331S/N333T/S408N/A436T 170 15L72N/A99G/H102W/E103W/V104H/R107P/Y112K/I183P/F249W/A282P/F306W/ His NOI331S/N333T/S408N/A436T 171 14L72N/A99G/H102W/E103W/V104F/R107P/G112Y/I183P/A282P/F306W/I331S/ His NON333T/S408N/A436T 172 15L72N/A99G/H102W/E103W/V104H/R107P/G112Y/I183P/A282P/F306W/I331S/ His NON333T/S408N/A436T AND Arginine Insertion between P107 and P108 173 16L72N/A99G/H102W/E103W/V104H/R107P/P108R/G112Y/I183P/A282P/F306W/ His NOI331S/N333T/S408N/A436T -AND Leucine insertion between P107 and R108 17414 L72N/A99G/H102W/E103W/V104H/R107P/Y112K/I183P/A282P/F306W/I331S/ HisNO N333T/S408N/A436T 175 15L72N/A99G/H102W/E103W/V104H/R107P/G112Y/I183P/K191M/A282P/F306W/ His NOI331S/N333T/S408N/A436T 176 17L72N/A99G/H102W/E103W/V104H/R107P/G112Y/I183P/M189I/I201S/N232S/ His NOA282P/F306W/I331S/N333T/S408N/A436T 177 18L72N/A99G/H102W/E103W/V104H/R107P/G112Y/I183P/E184A/M189I/K191R/ His NOI201F/A282P/F306W/I331S/N333T/S408N/A436T 178 17F71M/L72N/E103F/V104H/G105T/I110T/G112T/I183P/E184A/M187L/M189I/ His NOK191N/E197T/I201T/A280S/A282P/F306Y 179 22I183P/E184A/M187L/M189I/K191S/E197T/I201T/N375A/Y376C/K378G/K380G/ HisNO A382G/K384G/P386S/N411R/D413S/Q416T/E419L/K427M/N429E/G432A/A436T 18019 A99I/G112S/F306Y/I405L/S408N/A436T/I183P/E185T/M189I/K191N/N375H/ HisNO Y376L/K378P/K380S/A382R/K384N/P386K/V387L/N389E 181 13L72N/A99R/H102W/E103R/V104D/G112M/I183P/A282P/F306W/I331S/N333T/ His NOS408N/A436T 182 16L72N/A99G/H102W/E103W/V104H/R107P/G112M/I183P/I201F/F249W/A282P/ His NOF306W/I331S/N333T/S408N/A436T 183 17L72N/A99G/H102W/E103W/V104H/R107P/G112Y/I183P/M189I/I201C/F249W/ His NOA282P/F306W/I331S/N333T/S408N/A436T 184 15L72N/A99R/H102W/E103R/V104H/R107P/G112Y/I183P/A282P/F306W/I331S/ His NON333T/L337T/S408N/A436T 185 17L72N/A99R/H102W/E103R/V104H/R107P/I110A/G112Y/I183P/A280G/A282P/ His NOF306W/I331S/N333T/L337T/S408N/A436T

IV. Enzymatic Kynurenine Degradation for Therapy

In certain aspects, the polypeptides may be used for the treatment ofdiseases, including cancers that are sensitive to kynurenine depletion,with enzymes that deplete kynurenine, to prevent tumor-mediatedtolerogenic effects and instead mediate tumor-ablating pro-inflammatoryresponses. In certain aspects, kynureninases are contemplated for use intreating tumors expressing IDO1, IDO2, and/or TDO.

Certain aspects of the present invention provide a modified kynureninasefor treating diseases, such as tumors. Particularly, the modifiedpolypeptide may have human polypeptide sequences and thus may preventallergic reactions in human patients, allow repeated dosing, andincrease the therapeutic efficacy.

Tumors for which the present treatment methods are useful include anymalignant cell type, such as those found in a solid tumor or ahematological tumor. Exemplary solid tumors can include, but are notlimited to, a tumor of an organ selected from the group consisting ofpancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney,larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.Exemplary hematological tumors include tumors of the bone marrow, T or Bcell malignancies, leukemias, lymphomas, blastomas, myelomas, and thelike. Further examples of cancers that may be treated using the methodsprovided herein include, but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, leukemia, squamous cell cancer, lung cancer(including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung), cancerof the peritoneum, hepatocellular cancer, gastric or stomach cancer(including gastrointestinal cancer and gastrointestinal stromal cancer),pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, prostate cancer, vulval cancer, thyroid cancer, varioustypes of head and neck cancer, melanoma, superficial spreading melanoma,lentigo malignant melanoma, acral lentiginous melanomas, nodularmelanomas, as well as B-cell lymphoma (including low grade/follicularnon-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's macroglobulinemia), chroniclymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairycell leukemia, multiple myeloma, acute myeloid leukemia (AML) andchronic myeloblastic leukemia.

The cancer may specifically be of the following histological type,though it is not limited to these: neoplasm, malignant; carcinoma;carcinoma, undifferentiated; giant and spindle cell carcinoma; smallcell carcinoma; papillary carcinoma; squamous cell carcinoma;lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma;transitional cell carcinoma; papillary transitional cell carcinoma;adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cellcarcinoma; infiltrating duct carcinoma; medullary carcinoma; lobularcarcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma w/squamousmetaplasia; thymoma, malignant; ovarian stromal tumor, malignant;thecoma, malignant; granulosa cell tumor, malignant; androblastoma,malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipidcell tumor, malignant; paraganglioma, malignant; extra-mammaryparaganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignantmelanoma; amelanotic melanoma; superficial spreading melanoma; malignantmelanoma in giant pigmented nevus; epithelioid cell melanoma; bluenevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma,malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma;mixed tumor, malignant; mullerian mixed tumor; nephroblastoma;hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor,malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma,malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant;struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant;hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma;hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant;mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma;odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma,malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma;glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma;fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignantlymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse;malignant lymphoma, follicular; mycosis fungoides; other specifiednon-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mastcell sarcoma; immunoproliferative small intestinal disease; leukemia;lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcomacell leukemia; myeloid leukemia; basophilic leukemia; eosinophilicleukemia; monocytic leukemia; mast cell leukemia; megakaryoblasticleukemia; myeloid sarcoma; and hairy cell leukemia.

The kynureninase may be used herein as an antitumor agent in a varietyof modalities for depleting kynurenine and/or kynurenine-derivedmetabolites from tumor tissue, or the circulation of a mammal withcancer, or for depletion of kynurenine where its depletion is considereddesirable.

Depletion can be conducted in vivo in the circulation of a mammal, invitro in cases where kynurenine and/or kynurenine-derived metabolitesdepletion in tissue culture or other biological mediums is desired, andin ex vivo procedures where biological fluids, cells, or tissues aremanipulated outside the body and subsequently returned to the body ofthe patient mammal. Depletion of kynurenine from circulation, culturemedia, biological fluids, or cells is conducted to reduce the amount ofkynurenine accessible to the material being treated, and thereforecomprises contacting the material to be depleted with akynurenine-depleting amount of the kynureninase underkynurenine-depleting conditions as to degrade the ambient kynurenine inthe material being contacted.

The depletion may be directed to the nutrient source for the cells, andnot necessarily the cells themselves. Therefore, in an in vivoapplication, treating a tumor cell includes contacting the nutrientmedium for a population of tumor cells with the kynureninase. In thisembodiment, the medium may be blood, lymphatic fluid, spinal fluid andthe like bodily fluid where kynurenine depletion is desired.

Kynurenine- and/or kynurenine-derived metabolites depletion efficiencycan vary widely depending upon the application, and typically dependsupon the amount of kynurenine present in the material, the desired rateof depletion, and the tolerance of the material for exposure tokynureninase. Kynurenine and kynurenine metabolite levels in a material,and therefore rates of kynurenine and kynurenine metabolite depletionfrom the material, can readily be monitored by a variety of chemical andbiochemical methods well known in the art. Exemplarykynurenine-depleting amounts are described further herein, and can rangefrom 0.001 to 100 units (U) of kynureninase, preferably about 0.01 to 10U, and more preferably about 0.1 to 5 U kyureninase per milliliter (mL)of material to be treated. Typical dosages can be administered based onbody weight, and are in the range of about 5-1000 U/kilogram (kg)/day,preferably about 5-100 U/kg/day, more preferably about 10-50 U/kg/day,and more preferably about 20-40 U/kg/day.

Kynurenine-depleting conditions are buffer and temperature conditionscompatible with the biological activity of a kynureninase, and includemoderate temperature, salt, and pH conditions compatible with theenzyme, for example, physiological conditions. Exemplary conditionsinclude about 4-40° C., ionic strength equivalent to about 0.05 to 0.2 MNaCl, and a pH of about 5 to 9, while physiological conditions areincluded.

In a particular embodiment, the invention contemplates methods of usinga kynureninase as an antitumor agent, and therefore comprises contactinga population of tumor cells with a therapeutically effective amount ofkynureninase for a time period sufficient to inhibit tumor cell growth.

A therapeutically effective amount of a kynureninase is a predeterminedamount calculated to achieve the desired effect, i.e., to depletekynurenine in the tumor tissue or in a patient's circulation, andthereby mediate a tumor-ablating pro-inflammatory response. Thus, thedosage ranges for the administration of kynureninase of the inventionare those large enough to produce the desired effect in which thesymptoms of tumor cell division and cell cycling are reduced. The dosageshould not be so large as to cause adverse side effects, such ashyperviscosity syndromes, pulmonary edema, congestive heart failure,neurological effects, and the like. Generally, the dosage will vary withage of, condition of, sex of, and extent of the disease in the patientand can be determined by one of skill in the art. The dosage can beadjusted by the individual physician in the event of any complication.

The kynureninase can be administered parenterally by injection or bygradual infusion over time. The kynureninase can be administeredintravenously, intraperitoneally, orally, intramuscularly,subcutaneously, intracavity, transdermally, dermally, can be deliveredby peristaltic means, can be injected directly into the tissuecontaining the tumor cells, or can be administered by a pump connectedto a catheter that may contain a potential biosensor for kynurenine.

The therapeutic compositions containing kynureninase are conventionallyadministered intravenously, as by injection of a unit dose, for example.The term “unit dose” when used in reference to a therapeutic compositionrefers to physically discrete units suitable as unitary dosage for thesubject, each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect inassociation with the required diluent, i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitionerand are peculiar to each individual. However, suitable dosage ranges forsystemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for initial administration and boostershots are also contemplated and are typified by an initialadministration followed by repeated doses at one or more hour intervalsby a subsequent injection or other administration. Exemplary multipleadministrations are described herein and are particularly preferred tomaintain continuously high serum and tissue levels of kynureninase andconversely low serum and tissue levels of kynurenine. Alternatively,continuous intravenous infusion sufficient to maintain concentrations inthe blood in the ranges specified for in vivo therapies arecontemplated.

V. Conjugates

Compositions and methods of the present invention involve modifiedkynureninases, such as by forming conjugates with heterologous peptidesegments or polymers, such as polyethylene glycol. In further aspects,the kynureninases may be linked to PEG to increase the hydrodynamicradius of the enzyme and hence increase the serum persistence. Incertain aspects, the disclosed polypeptide may be conjugated to anytargeting agent, such as a ligand having the ability to specifically andstably bind to an external receptor or binding site on a tumor cell(U.S. Patent Publ. 2009/0304666).

A. Fusion Proteins

Certain embodiments of the present invention concern fusion proteins.These molecules may have a native or modified kynureninase linked at theN- or C-terminus to a heterologous domain. For example, fusions may alsoemploy leader sequences from other species to permit the recombinantexpression of a protein in a heterologous host. Another useful fusionincludes the addition of a protein affinity tag, such as a serum albuminaffinity tag or six histidine residues, or an immunologically activedomain, such as an antibody epitope, preferably cleavable, to facilitatepurification of the fusion protein. Non-limiting affinity tags includepolyhistidine, chitin binding protein (CBP), maltose binding protein(MBP), and glutathione-S-transferase (GST).

In a particular embodiment, the kynureninase may be linked to a peptidethat increases the in vivo half-life, such as an XTEN polypeptide(Schellenberger et al., 2009), IgG Fc domain, albumin, or albuminbinding peptide.

Methods of generating fusion proteins are well known to those of skillin the art. Such proteins can be produced, for example, by de novosynthesis of the complete fusion protein, or by attachment of the DNAsequence encoding the heterologous domain, followed by expression of theintact fusion protein.

Production of fusion proteins that recover the functional activities ofthe parent proteins may be facilitated by connecting genes with abridging DNA segment encoding a peptide linker that is spliced betweenthe polypeptides connected in tandem. The linker would be of sufficientlength to allow proper folding of the resulting fusion protein.

B. PEGylation

In certain aspects of the invention, methods and compositions related toPEGylation of kynureninase are disclosed. For example, the kynureninasemay be PEGylated in accordance with the methods disclosed herein.

PEGylation is the process of covalent attachment of poly(ethyleneglycol) polymer chains to another molecule, normally a drug ortherapeutic protein. PEGylation is routinely achieved by incubation of areactive derivative of PEG with the target macromolecule. The covalentattachment of PEG to a drug or therapeutic protein can “mask” the agentfrom the host's immune system (reduced immunogenicity and antigenicity)or increase the hydrodynamic size (size in solution) of the agent, whichprolongs its circulatory time by reducing renal clearance. PEGylationcan also provide water solubility to hydrophobic drugs and proteins.

The first step of the PEGylation is the suitable functionalization ofthe PEG polymer at one or both terminals. PEGs that are activated ateach terminus with the same reactive moiety are known as“homobifunctional,” whereas if the functional groups present aredifferent, then the PEG derivative is referred as “heterobifunctional”or “heterofunctional.” The chemically active or activated derivatives ofthe PEG polymer are prepared to attach the PEG to the desired molecule.

The choice of the suitable functional group for the PEG derivative isbased on the type of available reactive group on the molecule that willbe coupled to the PEG. For proteins, typical reactive amino acidsinclude lysine, cysteine, histidine, arginine, aspartic acid, glutamicacid, serine, threonine, and tyrosine. The N-terminal amino group andthe C-terminal carboxylic acid can also be used.

The techniques used to form first generation PEG derivatives aregenerally reacting the PEG polymer with a group that is reactive withhydroxyl groups, typically anhydrides, acid chlorides, chloroformates,and carbonates. In the second generation PEGylation chemistry moreefficient functional groups, such as aldehyde, esters, amides, etc., aremade available for conjugation.

As applications of PEGylation have become more and more advanced andsophisticated, there has been an increase in need for heterobifunctionalPEGs for conjugation. These heterobifunctional PEGs are very useful inlinking two entities, where a hydrophilic, flexible, and biocompatiblespacer is needed. Preferred end groups for heterobifunctional PEGs aremaleimide, vinyl sulfones, pyridyl disulfide, amine, carboxylic acids,and NHS esters.

The most common modification agents, or linkers, are based on methoxyPEG (mPEG) molecules. Their activity depends on adding aprotein-modifying group to the alcohol end. In some instancespolyethylene glycol (PEG diol) is used as the precursor molecule. Thediol is subsequently modified at both ends in order to make a hetero- orhomo-dimeric PEG-linked molecule.

Proteins are generally PEGylated at nucleophilic sites, such asunprotonated thiols (cysteinyl residues) or amino groups. Examples ofcysteinyl-specific modification reagents include PEG maleimide, PEGiodoacetate, PEG thiols, and PEG vinylsulfone. All four are stronglycysteinyl-specific under mild conditions and neutral to slightlyalkaline pH but each has some drawbacks. The thioether formed with themaleimides can be somewhat unstable under alkaline conditions so theremay be some limitation to formulation options with this linker. Thecarbamothioate linkage formed with iodo PEGS is more stable, but freeiodine can modify tyrosine residues under some conditions. PEG thiolsform disulfide bonds with protein thiols, but this linkage can also beunstable under alkaline conditions. PEG-vinylsulfone reactivity isrelatively slow compared to maleimide and iodo PEG; however, thethioether linkage formed is quite stable. Its slower reaction rate alsocan make the PEG-vinylsulfone reaction easier to control.

Site-specific PEGylation at native cysteinyl residues is seldom carriedout, since these residues are usually in the form of disulfide bonds orare required for biological activity. On the other hand, site-directedmutagenesis can be used to incorporate cysteinyl PEGylation sites forthiol-specific linkers. The cysteine mutation must be designed such thatit is accessible to the PEGylation reagent and is still biologicallyactive after PEGylation.

Amine-specific modification agents include PEG NHS ester, PEG tresylate,PEG aldehyde, PEG isothiocyanate, and several others. All react undermild conditions and are very specific for amino groups. The PEG NHSester is probably one of the more reactive agents; however, its highreactivity can make the PEGylation reaction difficult to control on alarge scale. PEG aldehyde forms an imine with the amino group, which isthen reduced to a secondary amine with sodium cyanoborohydride. Unlikesodium borohydride, sodium cyanoborohydride will not reduce disulfidebonds. However, this chemical is highly toxic and must be handledcautiously, particularly at lower pH where it becomes volatile.

Due to the multiple lysine residues on most proteins, site-specificPEGylation can be a challenge. Fortunately, because these reagents reactwith unprotonated amino groups, it is possible to direct the PEGylationto lower-pK amino groups by performing the reaction at a lower pH.Generally the pK of the alpha-amino group is 1-2 pH units lower than theepsilon-amino group of lysine residues. By PEGylating the molecule at pH7 or below, high selectivity for the N-terminus frequently can beattained. However, this is only feasible if the N-terminal portion ofthe protein is not required for biological activity. Still, thepharmacokinetic benefits from PEGylation frequently outweigh asignificant loss of in vitro bioactivity, resulting in a product withmuch greater in vivo bioactivity regardless of PEGylation chemistry.

There are several parameters to consider when developing a PEGylationprocedure. Fortunately, there are usually no more than four or five keyparameters. The “design of experiments” approach to optimization ofPEGylation conditions can be very useful. For thiol-specific PEGylationreactions, parameters to consider include: protein concentration,PEG-to-protein ratio (on a molar basis), temperature, pH, reaction time,and in some instances, the exclusion of oxygen. (Oxygen can contributeto intermolecular disulfide formation by the protein, which will reducethe yield of the PEGylated product.) The same factors should beconsidered (with the exception of oxygen) for amine-specificmodification except that pH may be even more critical, particularly whentargeting the N-terminal amino group.

For both amine- and thiol-specific modifications, the reactionconditions may affect the stability of the protein. This may limit thetemperature, protein concentration, and pH. In addition, the reactivityof the PEG linker should be known before starting the PEGylationreaction. For example, if the PEGylation agent is only 70 percentactive, the amount of PEG used should ensure that only active PEGmolecules are counted in the protein-to-PEG reaction stoichiometry.

VI. Proteins and Peptides

In certain embodiments, the present invention concerns novelcompositions comprising at least one protein or peptide, such as akynureninase. These peptides may be comprised in a fusion protein orconjugated to an agent as described supra.

As used herein, a protein or peptide generally refers, but is notlimited to, a protein of greater than about 200 amino acids, up to afull length sequence translated from a gene; a polypeptide of greaterthan about 100 amino acids; and/or a peptide of from about 3 to about100 amino acids. For convenience, the terms “protein,” “polypeptide,”and “peptide” are used interchangeably herein.

As used herein, an “amino acid residue” refers to any naturallyoccurring amino acid, any amino acid derivative, or any amino acid mimicknown in the art. In certain embodiments, the residues of the protein orpeptide are sequential, without any non-amino acids interrupting thesequence of amino acid residues. In other embodiments, the sequence maycomprise one or more non-amino acid moieties. In particular embodiments,the sequence of residues of the protein or peptide may be interrupted byone or more non-amino acid moieties.

Accordingly, the term “protein or peptide” encompasses amino acidsequences comprising at least one of the 20 common amino acids found innaturally occurring proteins, or at least one modified or unusual aminoacid.

Proteins or peptides may be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides, orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, or the chemical synthesisof proteins or peptides. The nucleotide and protein, polypeptide, andpeptide sequences corresponding to various genes have been previouslydisclosed, and may be found at computerized databases known to those ofordinary skill in the art. One such database is the National Center forBiotechnology Information's Genbank and GenPept databases (available onthe world wide web at ncbi.nlm.nih.gov/). The coding regions for knowngenes may be amplified and/or expressed using the techniques disclosedherein or as would be known to those of ordinary skill in the art.Alternatively, various commercial preparations of proteins,polypeptides, and peptides are known to those of skill in the art.

VII. Nucleic Acids and Vectors

In certain aspects of the invention, nucleic acid sequences encoding akynureninase or a fusion protein containing a kynureninase may bedisclosed. Depending on which expression system is used, nucleic acidsequences can be selected based on conventional methods. For example, ifthe kynureninase is derived from human kynureninase and containsmultiple codons that are rarely utilized in E. coli, then that mayinterfere with expression. Therefore, the respective genes or variantsthereof may be codon optimized for E. coli expression. Various vectorsmay be also used to express the protein of interest. Exemplary vectorsinclude, but are not limited, plasmid vectors, viral vectors,transposon, or liposome-based vectors.

VIII. Host Cells

Host cells may be any that may be transformed to allow the expressionand secretion of kynureninase and conjugates thereof. The host cells maybe bacteria, mammalian cells, yeast, or filamentous fungi. Variousbacteria include Escherichia and Bacillus. Yeasts belonging to thegenera Saccharomyces, or Pichia would find use as an appropriate

IX. Pharmaceutical Compositions

It is contemplated that the novel kynureninase can be administeredsystemically or locally to inhibit tumor cell growth and, mostpreferably, to kill cancer cells in cancer patients with locallyadvanced or metastatic cancers. They can be administered intravenously,intrathecally, and/or intraperitoneally. They can be administered aloneor in combination with anti-proliferative drugs. In one embodiment, theyare administered to reduce the cancer load in the patient prior tosurgery or other procedures. Alternatively, they can be administeredafter surgery to ensure that any remaining cancer (e.g., cancer that thesurgery failed to eliminate) does not survive.

It is not intended that the present invention be limited by theparticular nature of the therapeutic preparation. For example, suchcompositions can be provided in formulations together withphysiologically tolerable liquid, gel, or solid carriers, diluents, andexcipients. These therapeutic preparations can be administered tomammals for veterinary use, such as with domestic animals, and clinicaluse in humans in a manner similar to other therapeutic agents. Ingeneral, the dosage required for therapeutic efficacy will varyaccording to the type of use and mode of administration, as well as theparticularized requirements of individual subjects.

Such compositions are typically prepared as liquid solutions orsuspensions, as injectables. Suitable diluents and excipients are, forexample, water, saline, dextrose, glycerol, or the like, andcombinations thereof. In addition, if desired, the compositions maycontain minor amounts of auxiliary substances, such as wetting oremulsifying agents, stabilizing agents, or pH buffering agents.

Where clinical applications are contemplated, it may be necessary toprepare pharmaceutical compositions comprising proteins, antibodies, anddrugs in a form appropriate for the intended application. Generally,pharmaceutical compositions may comprise an effective amount of one ormore kynureninase or additional agents dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic, or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a pharmaceutical composition thatcontains at least one kyureninase isolated by the method disclosedherein, or additional active ingredient will be known to those of skillin the art in light of the present disclosure, as exemplified byRemington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated hereinby reference. Moreover, for animal (e.g., human) administration, it willbe understood that preparations should meet sterility, pyrogenicity,general safety, and purity standards as required by the FDA Office ofBiological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed., 1990, incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the pharmaceutical compositions is contemplated.

Certain embodiments of the present invention may comprise differenttypes of carriers depending on whether it is to be administered insolid, liquid, or aerosol form, and whether it needs to be sterile forthe route of administration, such as injection. The compositions can beadministered intravenously, intradermally, transdermally, intrathecally,intraarterially, intraperitoneally, intranasally, intravaginally,intrarectally, intramuscularly, subcutaneously, mucosally, orally,topically, locally, by inhalation (e.g., aerosol inhalation), byinjection, by infusion, by continuous infusion, by localized perfusionbathing target cells directly, via a catheter, via a lavage, in lipidcompositions (e.g., liposomes), or by other methods or any combinationof the forgoing as would be known to one of ordinary skill in the art(see, for example, Remington's Pharmaceutical Sciences, 18th Ed., 1990,incorporated herein by reference).

The modified polypeptides may be formulated into a composition in a freebase, neutral, or salt form. Pharmaceutically acceptable salts includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganicacids, such as, for example, hydrochloric or phosphoric acids, or suchorganic acids as acetic, oxalic, tartaric, or mandelic acid. Saltsformed with the free carboxyl groups can also be derived from inorganicbases, such as, for example, sodium, potassium, ammonium, calcium, orferric hydroxides; or such organic bases as isopropylamine,trimethylamine, histidine, or procaine. Upon formulation, solutions willbe administered in a manner compatible with the dosage formulation andin such amount as is therapeutically effective. The formulations areeasily administered in a variety of dosage forms, such as formulated forparenteral administrations, such as injectable solutions, or aerosolsfor delivery to the lungs, or formulated for alimentary administrations,such as drug release capsules and the like.

Further in accordance with certain aspects of the present invention, thecomposition suitable for administration may be provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent, or carrier is detrimental to the recipient or to thetherapeutic effectiveness of the composition contained therein, its usein administrable composition for use in practicing the methods isappropriate. Examples of carriers or diluents include fats, oils, water,saline solutions, lipids, liposomes, resins, binders, fillers, and thelike, or combinations thereof. The composition may also comprise variousantioxidants to retard oxidation of one or more component. Additionally,the prevention of the action of microorganisms can be brought about bypreservatives, such as various antibacterial and antifungal agents,including but not limited to parabens (e.g., methylparabens,propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal orcombinations thereof.

In accordance with certain aspects of the present invention, thecomposition is combined with the carrier in any convenient and practicalmanner, i.e., by solution, suspension, emulsification, admixture,encapsulation, absorption, and the like. Such procedures are routine forthose skilled in the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner, such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in acomposition include buffers, amino acids, such as glycine and lysine,carbohydrates, such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle composition that includes kynureninases,one or more lipids, and an aqueous solvent. As used herein, the term“lipid” will be defined to include any of a broad range of substancesthat is characteristically insoluble in water and extractable with anorganic solvent. This broad class of compounds is well known to those ofskill in the art, and as the term “lipid” is used herein, it is notlimited to any particular structure. Examples include compounds thatcontain long-chain aliphatic hydrocarbons and their derivatives. A lipidmay be naturally occurring or synthetic (i.e., designed or produced byman). However, a lipid is usually a biological substance. Biologicallipids are well known in the art, and include for example, neutral fats,phospholipids, phosphoglycerides, steroids, terpenes, lysolipids,glycosphingolipids, glycolipids, sulphatides, lipids with ether- andester-linked fatty acids, polymerizable lipids, and combinationsthereof. Of course, compounds other than those specifically describedherein that are understood by one of skill in the art as lipids are alsoencompassed by the compositions and methods.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the kynureninase or a fusion protein thereof maybe dispersed in a solution containing a lipid, dissolved with a lipid,emulsified with a lipid, mixed with a lipid, combined with a lipid,covalently bonded to a lipid, contained as a suspension in a lipid,contained or complexed with a micelle or liposome, or otherwiseassociated with a lipid or lipid structure by any means known to thoseof ordinary skill in the art. The dispersion may or may not result inthe formation of liposomes.

The actual dosage amount of a composition administered to an animalpatient can be determined by physical and physiological factors, such asbody weight, severity of condition, the type of disease being treated,previous or concurrent therapeutic interventions, idiopathy of thepatient, and on the route of administration. Depending upon the dosageand the route of administration, the number of administrations of apreferred dosage and/or an effective amount may vary according to theresponse of the subject. The practitioner responsible for administrationwill, in any event, determine the concentration of active ingredient(s)in a composition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, an active compound may comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared in such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors, such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations, will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 milligram/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 milligram/kg/body weightto about 100 milligram/kg/body weight, about 5 microgram/kg/body weightto about 500 milligram/kg/body weight, etc., can be administered, basedon the numbers described above.

X. Combination Treatments

In certain embodiments, the compositions and methods of the presentembodiments involve administration of a kynureninase in combination witha second or additional therapy. Such therapy can be applied in thetreatment of any disease that is associated with kynurenine dependency.For example, the disease may be cancer.

The methods and compositions, including combination therapies, enhancethe therapeutic or protective effect, and/or increase the therapeuticeffect of another anti-cancer or anti-hyperproliferative therapy.Therapeutic and prophylactic methods and compositions can be provided ina combined amount effective to achieve the desired effect, such as thekilling of a cancer cell and/or the inhibition of cellularhyperproliferation. This process may involve administering akynureninase and a second therapy. The second therapy may or may nothave a direct cytotoxic effect. For example, the second therapy may bean agent that upregulates the immune system without having a directcytotoxic effect. A tissue, tumor, or cell can be exposed to one or morecompositions or pharmacological formulation(s) comprising one or more ofthe agents (e.g., a kynureninase or an anti-cancer agent), or byexposing the tissue, tumor, and/or cell with two or more distinctcompositions or formulations, wherein one composition provides 1) akynureninase, 2) an anti-cancer agent, or 3) both a kynureninase and ananti-cancer agent. Also, it is contemplated that such a combinationtherapy can be used in conjunction with chemotherapy, radiotherapy,surgical therapy, or immunotherapy.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing, for example, both agents are delivered to a cellin a combined amount effective to kill the cell or prevent it fromdividing.

A kynureninase may be administered before, during, after, or in variouscombinations relative to an anti-cancer treatment. The administrationsmay be in intervals ranging from concurrently to minutes to days toweeks. In embodiments where the kynureninase is provided to a patientseparately from an anti-cancer agent, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the two compounds would still be able to exert anadvantageously combined effect on the patient. In such instances, it iscontemplated that one may provide a patient with the kynureninase andthe anti-cancer therapy within about 12 to 24 or 72 h of each other and,more particularly, within about 6-12 h of each other. In some situationsit may be desirable to extend the time period for treatmentsignificantly where several days (2, 3, 4, 5, 6, or 7) to several weeks(1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.

In certain embodiments, a course of treatment will last 1-90 days ormore (this such range includes intervening days). It is contemplatedthat one agent may be given on any day of day 1 to day 90 (this suchrange includes intervening days) or any combination thereof, and anotheragent is given on any day of day 1 to day 90 (this such range includesintervening days) or any combination thereof. Within a single day(24-hour period), the patient may be given one or multipleadministrations of the agent(s). Moreover, after a course of treatment,it is contemplated that there is a period of time at which noanti-cancer treatment is administered. This time period may last 1-7days, and/or 1-5 weeks, and/or 1-12 months or more (this such rangeincludes intervening days), depending on the condition of the patient,such as their prognosis, strength, health, etc. It is expected that thetreatment cycles would be repeated as necessary.

Various combinations may be employed. For the example below akynureninase is “A” and an anti-cancer therapy is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of any compound or therapy of the present embodiments toa patient will follow general protocols for the administration of suchcompounds, taking into account the toxicity, if any, of the agents.Therefore, in some embodiments there is a step of monitoring toxicitythat is attributable to combination therapy.

XI. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1—Development and Characterization of Human Kynase (h-KYNase)

The present studies involved engineering h-KYNase variants and developthe variants as therapeutics for a wide range of tumors. Numerous humanvariants were engineered and their catalytic activity (K_(cat)/K_(m))for Kyn, comparable to bacterial tool Kynases, was determined (FIG. 1Aand Table 2, below). The variants were optimized for increased catalyticactivity and for resistance to deactivation in 90% human serum in vitro(expressed as residual activity following incubation in serum for acertain time). It was found that the engineered human variants vastlyimproved catalytic stability in human serum in vitro (FIG. 1B and Table2).

The human variants, such as proteins (68) and (153), showed prolongedKyn degradation in mice after a single intravenous dose (FIG. 1C). Forthese studies BALB/c female mice, 6-8 weeks, weighed approximately 18-20g, were dosed iv with either vehicle (PBS pH7.4) or 10 mg/kg (68)/(153).At 6, 24, 48, 72 & 120 hrs after dosing, mice were terminally bled (n=4mice/time point) and the plasma samples were immediately quenched withacid containing Internal Standard (IS) solution and processed for LC/MSanalysis of Kynurenine.

TABLE 2 Catalytic activities and serum stability for selected enzymevariants Co- Fold over Stability in Average % Stability Protein factorWild Type k_(cat) (s⁻¹) K_(m) (mM) k_(cat)/K_(m) (M⁻¹ s⁻¹) 90% Serum 24hr 48 hr 72 hr 120 hr 2 PLP 27 ± 9 3 PLP 28 0.86 0.31 2800 4 PLP  19 ±10 5 PLP 15 0.85 0.57 1500 6 PLP 16 ± 6 7 PLP 12 0.71 0.57 1246 8 PLP 15± 5 9 PLP 10 0.71 0.69 1023 10 PLP  58 ± 12 11 PLP 176 0.6 0.034 1764712 PLP 554 1.21 0.022 55367 13 PLP 698 1.5 0.021 69775 14 PLP 24 1 0.432350 15 PLP 20 1.22 0.67 1967 16 PLP 29 1.4 0.48 2920 17 PLP 23 1.680.75 2250 18 PLP 20 1.38 0.7 2000 19 PLP 587 0.55 0.0093 58700 20 PLP388 0.993 0.026 38789 21 PLP 424 0.94 0.022 42398 22 PLP 342 1.06 0.03134226 23 PLP 405 0.85 0.021 40524 24 PLP 379 0.87 0.023 37913 25 PLP 3360.7 0.021 33623 26 PLP 389 0.65 0.017 38916 27 PLP 368 0.76 0.021 3682928 PLP 305 1.109 0.036 30467 29 PLP 526 1.32 0.0251 52590 30 PLP 3421.06 0.031 34226 31 PLP 277 0.698 0.0252 27698 32 PLP 550 0.852 0.015554968 33 PLP 580 1.3 0.022 57979 34 PLP 366 1.011 0.0276 36630 35 PLP219 0.874 0.0399 21904 36 PLP 270 0.645 0.0239 26987 37 PLP 536 0.6380.0119 53613 38 PLP 488 0.84 0.0173 48790 39 PLP 90 0.53 0.06 9020 40PLP 24 0.22 0.09 2400 41 PLP 2.1 0.50 1.90 260 42 PLP 4 0.250 0.640 40043 PLP 4 0.30 0.70 428 44 PLP 3 0.15 0.48 313 45 PLP 3 0.270 0.950 28046 PLP 3 0.250 1.000 250 47 PLP 3 0.250 0.730 335 48 PLP 3 0.320 0.950340 49 PLP 3 0.27 0.8 340 50 PLP 3 0.23 0.7 320 51 PLP 52 PLP 53 PLP 5910.975 0.017 59075 54 PLP 457 1.737 0.038 45706 55 PLP 512 1.024 0.02051222 56 PLP 269 1.042 0.039 26861 57 PLP 363 0.970 0.027 36330 58 PLP411 1.192 0.029 41111 59 PLP 635 1.098 0.017 63492 60 PLP 788 1.0480.013 78780 61 PLP 368 0.805 0.022 36770 62 PLP 359 0.507 0.014 35933 63PLP 415 0.954 0.023 41464 64 PLP 502 0.457 0.009 50183 65 PLP 100 0.2620.026 10046 66 PLP 127 0.253 0.020 12666 67 PLP 47 0.210 0.044 4729 68PLP 96 0.471 0.049 9554  41 ± 11 28 ± 8 14 ± 2 69 PLP 127 0.580 0.04512692 70 PLP 70 0.470 0.067 7015 71 PLP 0.94 0.089 10506 72 PLP 2.150.14 15357 73 PLP 242 1.7 70 24200 74 PLP 75 PLP 76 PLP 77 PLP 78 PLP 79PLP 80 PLP 81 PLP 82 PLP 110 0.87 0.0787 11054.63787 83 PLP 159 1.270.08 15875 84 PLP 292 1.75 0.06 29167 85 PLP 85 1.27 0.15 8467 86 PLP149 1.28 0.086 14884 87 PLP 100 0.712 0.071 10028 88 PLP 63 0.95 0.156333 89 PLP 138 1.38 0.1 13800 90 PLP 120 1.2 0.1 12000 91 PLP 141 0.820.058 14137.93103 92 PLP 90 0.72 0.08 9000 93 PLP 83 1 0.12 8333 94 PLP200 1.2 0.06 20000 95 PLP 96 PLP 137 1.5 0.1 13700 97 PLP 67 0.67 0.0996729 98 PLP 0.58 0.118 4897 99 PLP 0.73 0.089 8266 100 PLP 0.73 0.04616148 101 PLP 1.02 0.055 18569 102 PLP 0.97 0.087 11141 103 PLP 0.920.041 22282 104 PLP 0.96 0.049 19537 105 PLP 1.01 0.053 18825 106 PLP145 0.93 0.065 14470 107 PLP 0.71 0.037 19165 108 PLP 0.47 0.119 3981109 PLP 0.81 0.029 27722 110 PLP 0.99 0.039 25306 111 PLP 135.1 1.180.09 13511 112 PLP 140.2 0.76 0.05 14016 113 PLP 248 1.44 0.06 24798 9 3114 PLP 271.7 0.89 0.032 27169 115 PLP 68.8 0.67 0.1 6883 116 PLP 173.31 0.06 17326 117 PLP 132.3 0.99 0.08 13228 118 PLP 193.3 1.11 0.06 1933216 4 119 PLP 142 1.31 0.092 14205 120 PLP 108 0.81 0.075 10814 121 PLP197 0.42 0.021 19719 122 PLP 348 0.82 0.024 34797 123 PLP 81 1.03 0.1278106 124 PLP 244 0.66 0.027 24395 125 PLP 301 1.31 0.044 30103 126 PLP220 0.98 0.045 21971 127 PLP 315 1.26 0.040 31455 128 PLP 267 1.07 0.04026684 129 PLP 193 0.86 0.045 19309 130 PLP 169 0.58 0.034 16898 131 PLP309 1.10 0.036 30903 132 PLP 192 1.15 0.060 19201 133 PLP 169 1.07 0.06316870 134 PLP 150 0.82 0.054 15035 135 PLP 75 0.82 0.109 7487 136 PLP350 1.235 0.035 35286 137 PLP 210 0.943 0.0449 21002 4 2 1 138 PLP 3141.414 0.045 31422 45 ± 8 27 ± 7 22 ± 4 139 PLP 243 1.418 0.0583 24322140 PLP 181 0.91 0.05 18068 141 PLP 160 0.97 0.06 16020 142 PLP 165 1.100.07 16467.07 33 18 2 143 PLP 159 1.07 0.07 15952.38 144 PLP 139 0.080.05 1400 145 PLP 88 0.93 0.11 8837.76 146 PLP 106 0.74 0.07 10584 147PLP 127 1.09 0.09 12740 148 PLP 144 1.06 0.07 14403.79 149 PLP 130 1.030.08 13039.09 150 PLP 130 1.03 0.05 18889 151 PLP 130 1.08 0.08 13626152 PLP 92 0.692 0.0756 9153 62 42 32 153 PLP 337 1.01 0.03 33667 72 ± 8 68 ± 10 56 ± 8 154 PLP 235 0.598 0.0255 23451 69 53 155 PLP 236 1.210.051 23633 26 5 0 156 PLP 353 1.15 0.033 35323 35 3 0 157 PLP 423 1.260.030 42282 76 59 38 158 PLP 309 1.14 0.037 30894 66 48 33 159 PLP 2961.39 0.047 29638 55 35 22 160 PLP 243 1.98 0.081 24335 41 16 3 161 PLP251 1.05 0.042 25120 71 66 55 162 PLP 248 1.12 0.045 24800 63 55 41 163PLP 173 0.99 0.058 17290 77 66 45 164 PLP 330 0.34 0.010 33010 16 5 0165 PLP 354 1.63 0.046 35430 87 51 42 166 PLP 347 1.06 0.031 34710 76 5343 167 PLP 316 1.04 0.033 31650 69 51 45 168 PLP 330 0.78 0.024 33000 97± 1  88 ± 11 82 ± 1 169 PLP 99 0.47 0.047 9900 73 75 56 170 PLP 177 0.700.040 17663.0 76 65 60 171 PLP 275 0.71 0.026 27517.0 65 63 57 172 PLP148 0.53 0.036 14750.0 79 79 65 173 PLP 209 0.99 0.047 20930.0 83 64 72174 PLP 274 0.58 0.021 27371.0 68 76 47 175 PLP 146 0.56 0.038 14605.069 73 65 176 PLP 206 0.60 0.029 20552.0 64 67 44 177 PLP 167 0.63 0.03816684.0 65 45 43 178 PLP 160 1.40 0.090 15500 179 PLP 14 1.10 0.80 1400180 PLP 20 1.40 0.70 2000 181 PLP 115 0.723 0.063 11476.2 70 22 182 PLP171 0.685 0.040 17125.0 52 25 183 PLP 171 0.684 0.040 17100.0 91 45 184PLP 346 1.440 0.042 34615.4 63 3 185 PLP 137 0.959 0.070 13700.0 0 0

Example 2—HsKYN Efficacy and Anti-Tumor Memory

Next, the in vivo efficacy of the HsKYN variant was tested in the CT26mouse model as well as B16-IDO models. 6-8 week old female BALB/c micewere implanted subcutaneously with 500,000 CT26 cells in 100 μl PBS intothe lower right flank. Dosing commenced on Day 4 after cell inoculation.Protein (68) was dosed subcutaneously at 10 mg/kg Q6D for 3 doses;Epacadostat was dosed orally at 300 mg/kg QD (5 days on 2 days off);AntiPD-1 was dosed intraperitonially at 10 mg/kg Q3D for 5 doses. Tumorswere measured three times a week with digital calipers and tumor volumeswere calculated using the formula Volume=(L×W×T)×Pi/6. Animals weresacrificed when tumors reach >2,000 mm³(FIG. 2A). One hundred days afterthe appearance of the last complete responder (CR) from (A), naïveBALB/c mice and CRs were re-challenged with CT26 implantedsubcutaneously into the lower left flank. Tumor volumes were measured asdescribed above and results are shown in FIG. 2B.

Example 3—HsKYN Tumor Immunoprofiling

Immunoprofiling of (68) was performed in CT26 tumor bearing mice. 6-8week old female BALB/c were implanted subcutaneously with 500,000 CT26cells in 100 μl PBS into the lower right flank. Dosing commenced on Day4 after cell inoculation. Tumors were measured three times a week withdigital calipers and tumor volumes were calculated using the formulaVolume=(L×W×T)×Pi/6. Animals were sacrificed 24h after the last dose andtumors collected for further analysis. Protein (68) was dosedsubcutaneously at 10 mg/kg Q6D for 2 doses; AntiPD-1 was dosedintraperitonially at 10 mg/kg Q3D for 3 doses. RNA was extracted usingQiagen RNAeasy kit and 100 ng of RNA were used for Nanostring analysisusing the PanCancer Immune Profiling Panel (FIG. 3A). For the studies inFIG. 3B, (68) was dosed subcutaneously at 10 mg/kg Q6D for 2 doses.Tumors were digested with the tumor dissociation kit from Miltenyi.Single cell suspensions were stained with labeled antibodies andanalyzed using Fortessa: CD8 (CD45+ CD3+ CD8+ CD4−); Tregs (CD45+CD3+CD4+ CD8− FoxP3+ CD25+); M1 macrophages (CD45+ CD11b+ F4/80+ MHCII+CD206−); M2 macrophages (CD45+ CD11b+ F4/80+ MHCII+ CD206+).

Example 4—Lead HsKYN Variant PD and PK Profiles in Rat and NHP

Male Sprague Dawley rats (200-300 g) were dosed iv, with either a singledose or 3 weekly doses of 10 mg/kg (153), by slow injection via tailvein. Blood was collected 24 hrs before dose (−24h), 6, 24, 48, 72 or120 hrs after 1st, 2nd or 3rd dose (n=4 mice/time point). Plasma sampleswere processed as described in FIG. 1C and Kynurenine levels wereanalyzed by LC/MS (FIG. 4A). Next, one male and one female Cynomolgusmonkeys (≥2 years old) were dosed iv with a single dose of 10 mg/kg(153) by slow injection via the cephalic vein. Blood was collectedpredose (0 h), 0.25, 6, 24, 48, 72, 120, 168, 240 and 336 hrs postdosing. Plasma samples were divided into two parts: one part wasprocessed as described in FIG. 1C for Kynurenine LC/MS analysis (FIG.4B); the other part was subject to ELISA analysis of pegylated protein(anti-PEG antibody (Abcam # ab51257) capture and biotinylated anti-PEGantibody (Abcam # ab53449) detection). Plasma concentration versus timedata were analyzed by non-compartmental approaches using the WinNonlinsoftware program, and pharmacokinetic parameters with C0, Cmax, T1/2,AUC0-last, AUC0-inf were calculated.

Thus, the present studies showed that Kynurenine degradation by Kynaseis broadly applicable to both IDO and TDO expressing tumors. HsKYNprotein engineering achieved a more than 500-fold increase of catalyticactivity, and vastly improved catalytic stability, leading to robust anddurable Kyn depletion in vivo. The HsKYN variant demonstrated superiorsingle agent and PD1 combination efficacy as compared to Epacadostat, aswell as sustained anti-tumor memory. The increased IFNg related T-cellinflammatory signature gene expression and M1/M2 macrophage ratio arelikely the mechanisms underlying HsKYN+/−PD1 mediated tumor efficacy.Finally, the favorable PK/PD profiles in preclinical tox species supportfurther clinical development of the lead HsKYN variant.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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The disclosure is further described in the following numberedembodiments:

Embodiment 1

An isolated, modified human kynureninase enzyme, said modified enzymehaving an amino acid sequence that is at least 90% identical to a humankynureninase enzyme of SEQ ID NO: 1 and comprising at least onesubstitution selected from the group consisting of LBP, K38E, Y47L,I48F, K50Q, I51M, S60N, K64N, D65G, E66K, N67D, N67P, D67S, A68F, A68T,A68V, F71L, F71M, L72N, K84E, E88N, E89K, E89S, E90Q, D92E, K93N, K93T,A95H, A95Q, K96N, I97H, I97L, I97V, A98G, A99G, A99I, A99R, A99S, A99T,A99V, Y100N, Y100S, Y100T, G101A, H102W, E103F, E103H, E103N, E103Q,E103R, E103V, E103W, V104D, V104E, V104F, V104H, V104K, V104L, V104R,G105A, G105H, G105S, G105T, E106D, K106D, K106E, K106H, K106N, R107P,R107S, P108R, I110A, I110F, I110L, I110M, I110T, T111D, T111H, T111N,T111R, G112A, G112C, G112D, G112K, G112L, G112M, G112Q, G112R, G112S,G112T, G112Y, N127K, I131V, A132V, L133V, A136T, L137T, T138S, N140D,H142Q, Q14R, Y156H, K163T, D168E, H169R, Q175L, I183F, I183L, I183P,I183S, E184A, E184D, E184R, E184T, E184V, E185T, M187L, M189I, K191A,K191G, K191H, K191M, K191N, K191R, K191S, K191T, K191W, E197A, E197D,E197F, E197K, E197M, E197Q, E197S, E197T, E197V, I201C, I201E, I201F,I201H, I201L, I201S, I201T, I201V, H203K, L219M, L219W, F220L, V223I,F225Y, H230F, H230L, H230Y, N232S, Y246F, F249W, D250E, S274A, S274C,S274G, S274N, S274T, L278M, A280G, A280S, A280T, G281S, A282M, A282P,G284N, I285L, V303L, V303S, F306W, F306Y, S311N, K315E, D317E, D317K,I331C, I331L, I331N, I331S, I331T, I331V, N333T, P334N, P335T, L337T,L338A, L338Q, S341I, K373E, K373N, N375A, N375H, Y376C, Y376F, Y376L,K378G, K378P, K378Q, K378R, K380G, K380S, A382G, A382R, A382T, T383S,K384G, K384N, P386K, P386S, V387L, N389E, I405L, F407Y, S408D, S408N,N411R, D413S, D413V, Q416T, E419A, E419L, K420E, R421N, V424I, K427M,N429E, G432A, and A4361.

Embodiment 2

The enzyme of embodiment 1, wherein the amino acid sequence is at least90% identical to the amino acid sequence of any one of the polypeptidesof Table 1.

Embodiment 3

The enzyme of embodiment 1 or 2, comprising a substitution at A282, F306and/or F249.

Embodiment 4

The enzyme of any one of embodiments 1-3, comprising the F306Wsubstitution.

Embodiment 5

The enzyme of any one of embodiments 1-4, comprising the L72Nsubstitution.

Embodiment 6

The enzyme of any one of embodiments 1-5, comprising the H102W and/orN333T substitutions.

Embodiment 7

The enzyme of any one of embodiments 1-6, comprising a substitution atA99.

Embodiment 8

The enzyme of any one of embodiments 1-7, comprising a substitution atG112.

Embodiment 9

The enzyme of any one of embodiments 1-8, comprising a substitution atE103.

Embodiment 10

The enzyme of any one of embodiments 1-9, comprising a substitution atV104.

Embodiment 11

The enzyme of any one of embodiments 1-10, comprising a substitution atS408.

Embodiment 12

The enzyme of any one of embodiments 1-11, comprising the I183Psubstitution.

Embodiment 13

The enzyme of any one of embodiments 1-12, comprising the R107Psubstitution.

Embodiment 14

The enzyme of any one of embodiments 1-13, comprising the A436Tsubstitution.

Embodiment 15

The enzyme of any one of embodiments 1-14, comprising at least onesubstitution selected from L72N, H102W, A282P, F306W, I331S and N333T.

Embodiment 16

The enzyme of any one of embodiments 1-15, comprising at least two,three, four or five substitutions selected from L72N, H102W, A282P,F306W, I331S and N333T.

Embodiment 17

The enzyme of any one of embodiments 1-16, comprising the substitutionsL72N, H102W, A282P, F306W, I331S and N333T.

Embodiment 18

The enzyme of any one of embodiments 1-17, wherein the enzyme has acatalytic activity relative the KYN (kcat/kM) of at least 8000M⁻¹s⁻¹.

Embodiment 19

The enzyme of any one of embodiments 1-18, wherein the enzyme has acatalytic activity for kynurenine (KYN) (kcat/kM) of between about8000M⁻¹s⁻¹ and 40000 M⁻¹s⁻¹; 10000M⁻¹s⁻¹ and 40000 M⁻¹s⁻¹; 20000M⁻¹s⁻¹and 40000 M⁻¹s⁻¹; or 25000M⁻¹s⁻¹ and 35000 M⁻¹s⁻¹.

Embodiment 20

The enzyme of any one of embodiments 1-19, wherein the amino acidsequence is at least 90% identical to the amino acid sequence of SEQ IDNO: 2.

Embodiment 21

The enzyme of embodiment 20, wherein the amino acid sequence is at least95% identical to the amino acid sequence of SEQ ID NO: 2.

Embodiment 22

The enzyme of any one of embodiments 1-19, wherein the amino acidsequence is at least 90% identical to the amino acid sequence of SEQ IDNO: 3.

Embodiment 23

The enzyme of embodiment 22, wherein the amino acid sequence is at least95% identical to the amino acid sequence of SEQ ID NO: 3.

Embodiment 24

The enzyme of any one of embodiments 1-23, further comprising aheterologous peptide segment.

Embodiment 25

The enzyme of any one of embodiments 1-24, wherein the enzyme is coupledto polyethylene glycol (PEG).

Embodiment 26

The enzyme of embodiment 25, wherein the enzyme is coupled to PEG viaone or more Lys or Cys residues.

Embodiment 27

A nucleic acid comprising a nucleotide sequence encoding the enzyme ofany one of embodiments 1-26.

Embodiment 28

The nucleic acid of embodiment 27, wherein the nucleic acid is codonoptimized for expression in bacteria, fungus, insects, or mammals.

Embodiment 29

An expression vector comprising the nucleic acid of embodiment 27 or 28.

Embodiment 30

A host cell comprising the nucleic acid of embodiment 27 or 28 or theexpression vector of embodiment 29.

Embodiment 31

The host cell of embodiment 30, wherein the host cell is a bacterialcell, a fungal cell, an insect cell, or a mammalian cell.

Embodiment 32

The host cell of embodiment 31, wherein the host cell is a bacterialcell.

Embodiment 33

The host cell of embodiment 31, wherein the host cell is a mammaliancell, optionally wherein the mammalian cell is an immune effector cell.

Embodiment 34

The host cell of embodiment 33, wherein the immune effector cell is aNK-cell or a T-cell.

Embodiment 35

A pharmaceutical formulation comprising an enzyme in accordance with anyone of embodiments 1-26 in a pharmaceutically acceptable carrier.

Embodiment 36

A method of treating a subject having a tumor comprising administeringto the subject an effective amount of the enzyme of any one ofembodiments 1-26 or the formulation of embodiment 35.

Embodiment 37

A composition comprising an effective amount of the enzyme of any one ofembodiments 1-26 or the formulation of embodiment 35, for use in thetreatment a subject having a tumor.

Embodiment 38

The method of embodiment 36 or composition of embodiment 37, wherein thesubject has been identified as having an IDO1, IDO2, or TDO-expressingtumor.

Embodiment 39

The method of embodiment 36 or 38, or the composition of embodiment 37or 38, wherein the tumor is a solid tumor.

Embodiment 40

The method of embodiment 36 or 38, or the composition of embodiment 37or 38, wherein the tumor is a hematological tumor.

Embodiment 41

The method of any one of embodiments 36 and 38-40, or the composition ofany one of embodiments 37-40, wherein the subject is a human patient.

Embodiment 42

The method of any one of embodiments 36 and 38-41, or the composition ofany one of embodiments 37-41, wherein the enzyme or formulation isadministered intratumorally, intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intraocularly, intranasally, intravitreally, intravaginally,intrarectally, intramuscularly, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,orally, by inhalation, by injection, by infusion, by continuousinfusion, by localized perfusion bathing target cells directly, via acatheter, or via a lavage.

Embodiment 43

The method of any one of embodiments 36 and 38-42, further comprisingadministering at least a second anti-cancer therapy.

Embodiment 44

The method of embodiment 43, wherein the second anticancer therapy isradiation therapy, surgical therapy, an immunotherapy or a secondanticancer compound.

Embodiment 45

The method of embodiment 44, wherein the second anticancer compound isan immune checkpoint inhibitor.

Embodiment 46

The method of embodiment 44 or 45, wherein the second anticancercompound is an antibody.

Embodiment 47

The method of any one of embodiments 44-46, wherein the secondanticancer compound comprises an anti-PD1, anti-CTLA-4, or anti-PD-L1antibody.

Embodiment 48

The method of any one of embodiments 44-47, wherein the secondanticancer compound is an antibody-drug conjugate.

Embodiment 49

The method of any one of embodiments 44-48, wherein the immunotherapycomprises administering immune effector cells or an immunogeniccomposition.

Embodiment 50

The method of embodiment 49, wherein the immunogenic compositioncomprises cancer cell antigens.

Embodiment 51

The method of embodiment 49 or 50, wherein the immune effector cellscomprise NK-cells or T-cells.

Embodiment 52

The method of any one of embodiments 49-51, wherein the immune effectorcells comprise CAR T-cells.

Embodiment 53

A transgenic T cell comprising an expressed kynureninase enzymeaccording to any one of embodiments 1-26.

Embodiment 54

The cell of embodiment 53, further comprising an expressed chimericantigen T-cell receptor (CAR) or an engineered T-cell receptor (TCR).

Embodiment 55

The cell of embodiment 53 or 54, wherein the cell is a human T cell.

Embodiment 56

The cell of embodiment 54 or 55, wherein DNA encoding the CAR and thekynureninase is integrated into the genome of the cell.

Embodiment 57

The cell of any one of embodiments 54-56, wherein the CAR is targeted toa cancer-cell antigen.

Embodiment 58

The cell of embodiment 57, wherein the cancer-cell antigen is HER2,CD19, CD20, or GD2.

Embodiment 59

A method of providing a T-cell response in a human subject having atumor comprising administering an effective amount of transgenic cellsin accordance with any one of embodiments 53-58 to the subject.

Embodiment 60

A composition comprising an effective amount of transgenic cells inaccordance with any one of embodiments 53-58, for use in the treatment asubject having a tumor.

Embodiment 61

The method of embodiment 59 or the composition of embodiment 60, whereinthe transgenic cells are autologous.

Embodiment 62

The method of embodiment 59 or the composition of embodiment 60, whereinthe transgenic cells are heterologous.

Embodiment 63

Use of a kynureninase enzyme in accordance with any one of embodiments1-26, a nucleic acid in accordance with embodiment 27 or 28, or anexpression vector in accordance with embodiment 29, in the manufactureof a medicament for the treatment of a tumor.

Sequence appendix: SEQ ID NO: 1: wild type human KYNaseMEPSSLELPADTVQRIAAELKCHPTDERVALHLDEEDKLRHFRECFYIPKIQDLPPVDLSLVNKDENAIYFLGNSLGLQPKMVKTYLEEELDKWAKIAAYGHEVGKRPWITGDESIVGLMKDIVGANEKEIALMNALTVNLHLLMLSFFKPTPKRYKILLEAKAFPSDHYAIESQLQLHGLNIEESMRMIKPREGEETLRIEDILEVIEKEGDSIAVILFSGVHFYTGQHFNIPAITKAGQAKGCYVGFDLAHAVGNVELYLHDWGVDFACWCSYKYLNAGAGGIAGAFIHEKHAHTIKPALVGWFGHELSTRFKMDNKLQLIPGVCGFRISNPPILLVCSLHASLEIFKQATMKALRKKSVLLTGYLEYLIKHNYGKDKAATKKPVVNIITPSHVEERGCQLTITFSVPNKDVFQELEKRGVVCDKRNPNGIRVAPVPLYNSFHDVYKFTNLLTSILDSAETKN SEQ ID NO: 2 Protein 168:MEPSSLELPADTVQRIAAELKCHPTDERVALHLDEEDKLRHFRECFYIPKIQDLPPVDLSLVNKDENAIYFNGNSLGLQPKMVKTYLEEELDKWAKIARYGWRHGKPPWITYDESIVGLMKDIVGANEKEIALMNALTVNLHLLMLSFFKPTPKRYKILLEAKAFPSDHYAIESQLQLHGLNPEESMRMIKPREGEETLRIEDILEVIEKEGDSIAVILFSGVHFYTGQHFNIPAITKAGQAKGCYVGWDLAHAVGNVELYLHDWGVDFACWCSYKYLNAGPGGIAGAFIHEKHAHTIKPALVGWWGHELSTRFKMDNKLQLIPGVCGFRSSTPPILLVCSLHASLEIFKQATMKALRKKSVLLTGYLEYLIKHNYGKDKAATKKPVVNIITPSHVEERGCQLTITFNVPNKDVFQELEKRGVVCDKRNPNGIRVTPVPLYNSFHDVYKF TNLLTSILDSAETKN*SEQ ID NO: 3 Protein 153:MEPSSLELPADTVQRIAAELKCHPTDERVALHLDEEDKLRHFRECFYIPKIQDLPPVDLSLVNKDENAIYFNGNSLGLQPKMVKTYLEEELDKWAKIARYGWRHGKPPWITYDESIVGLMKDIVGANEKEIALMNALTVNLHLLMLSFFKPTPKRYKILLEAKAFPSDHYAIESQLQLHGLNPEESMRMIKPREGEETLRIEDILEVIEKEGDSIAVILFSGVHFYTGQHFNIPAITKAGQAKGCYVGFDLAHAVGNVELYLHDWGVDFACWCSYKYLNAGPGGIAGAFIHEKHAHTIKPALVGWWGHELSTRFKMDNKLQLIPGVCGFRSSTPPILLVCSLHASLEIFKQATMKALRKKSVLLTGYLEYLIKHNYGKDKAATKKPVVNIITPSHVEERGCQLTITFNVPNKDVFQELEKRGVVCDKRNPNGIRVTPVPLYNSFHDVYKF TNLLTSILDSAETKN*

What is claimed is:
 1. An isolated, modified human kynureninase enzyme,said modified enzyme having an amino acid sequence that is at least 90%identical to a human kynureninase enzyme of SEQ ID NO: 1 and comprisingat least one substitution selected from the group consisting of LBP,K38E, Y47L, I48F, K50Q, I51M, S60N, K64N, D65G, E66K, N67D, N67P, D67S,A68F, A68T, A68V, F71L, F71M, L72N, K84E, E88N, E89K, E89S, E90Q, D92E,K93N, K93T, A95H, A95Q, K96N, I97H, I97L, I97V, A98G, A99G, A99I, A99R,A99S, A99T, A99V, Y100N, Y100S, Y100T, G101A, H102W, E103F, E103H,E103N, E103Q, E103R, E103V, E103W, V104D, V104E, V104F, V104H, V104K,V104L, V104R, G105A, G105H, G105S, G105T, E106D, K106D, K106E, K106H,K106N, R107P, R107S, P108R, I110A, I110F, I110L, I110M, I110T, T111D,T111H, T111N, T111R, G112A, G112C, G112D, G112K, G112L, G112M, G112Q,G112R, G112S, G112T, G112Y, N127K, I131V, A132V, L133V, A136T, L137T,T138S, N140D, H142Q, Q14R, Y156H, K163T, D168E, H169R, Q175L, I183F,I183L, I183P, I183S, E184A, E184D, E184R, E184T, E184V, E185T, M187L,M189I, K191A, K191G, K191H, K191M, K191N, K191R, K191S, K191T, K191W,E197A, E197D, E197F, E197K, E197M, E197Q, E197S, E197T, E197V, I201C,I201E, I201F, I201H, I201L, I201S, I201T, I201V, H203K, L219M, L219W,F220L, V223I, F225Y, H230F, H230L, H230Y, N232S, Y246F, F249W, D250E,S274A, S274C, S274G, S274N, S274T, L278M, A280G, A280S, A280T, G281S,A282M, A282P, G284N, I285L, V303L, V303S, F306W, F306Y, S311N, K315E,D317E, D317K, I331C, I331L, I331N, I331S, I331T, I331V, N333T, P334N,P335T, L337T, L338A, L338Q, S341I, K373E, K373N, N375A, N375H, Y376C,Y376F, Y376L, K378G, K378P, K378Q, K378R, K380G, K380S, A382G, A382R,A382T, T383S, K384G, K384N, P386K, P386S, V387L, N389E, I405L, F407Y,S408D, S408N, N411R, D413S, D413V, Q416T, E419A, E419L, K420E, R421N,V424I, K427M, N429E, G432A, and A4361.
 2. The enzyme of claim 1, whereinthe amino acid sequence is at least 90% identical to the amino acidsequence of any one of the polypeptides of Table
 1. 3. The enzyme ofclaim 1, comprising: (a) a substitution at A282, F306 or F249; (b) theF306W substitution; (c) the L72N substitution; (d) the H102W and N333Tsubstitutions; (e) a substitution at A99; (f) a substitution at G112;(g) a substitution at E103; (h) a substitution at V104; (i) asubstitution at S408; (j) the I183P substitution; (k) the R107Psubstitution; and/or (1) the A436T substitution.
 4. The enzyme of claim1, comprising one or more substitutions selected from L72N, H102W,A282P, F306W, I331S and N333T.
 5. The enzyme of claim 1, wherein theenzyme has a catalytic activity for kynurenine (KYN) (kcat/kM) of fromabout 8000M⁻¹s⁻¹ to about 40000 M⁻¹s⁻¹.
 6. The enzyme of claim 1,wherein the amino acid sequence is at least 90% identical to the aminoacid sequence of SEQ ID NO:
 2. 7. The enzyme of claim 6, wherein theamino acid sequence is at least 95% identical to the amino acid sequenceof SEQ ID NO:
 2. 8. The enzyme of claim 1, further comprising aheterologous peptide segment.
 9. The enzyme of claim 1, wherein theenzyme is coupled to polyethylene glycol.
 10. A nucleic acid comprisinga nucleotide sequence encoding the enzyme of claim
 1. 11. An expressionvector comprising the nucleic acid of claim
 10. 12. A host cellcomprising the nucleic acid of claim
 10. 13. A pharmaceuticalformulation comprising an enzyme of claim 1 in a pharmaceuticallyacceptable carrier.
 14. A method of treating a subject having a tumorcomprising administering to the subject an effective amount of theenzyme of claim
 1. 15. The method of claim 14, wherein: (a) the subjecthas been identified as having an IDO1, IDO2, or TDO expressing tumor;(b) the tumor is a solid tumor or a hematological tumor; (c) the subjectis a human patient; (d) the enzyme or formulation is administeredintratumorally, intravenously, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostaticaly, intrapleurally, intratracheally, intraocularly,intranasally, intravitreally, intravaginally, intrarectally,intramuscularly, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, orally, by inhalation,by injection, by infusion, by continuous infusion, by localizedperfusion bathing target cells directly, via a catheter, or via alavage; and/or (e) the enzyme or formulation is administered with atleast a second anti-cancer therapy selected from the group consisting ofradiation therapy, surgical therapy, an immunotherapy and a secondanticancer compound.
 16. The method of claim 15, wherein the secondanticancer compound is an immune checkpoint inhibitor and/or wherein theimmunotherapy comprises administering immune effector cells or animmunogenic composition to the subject.
 17. The method of claim 16,wherein: (a) the immune checkpoint inhibitor comprises an antibody orantibody-drug conjugate; (b) the immune checkpoint inhibitor comprisesan anti-PD1, anti-CTLA-4, or anti-PD-L1 antibody; (c) the immunogeniccomposition comprises cancer cell antigens; and/or (d) the immuneeffector cells comprise NK-cells, T-cells, or CAR T-cells.
 18. Atransgenic T cell comprising an expressed kynureninase enzyme accordingto claim
 1. 19. The transgenic T cell of claim 19, wherein: (a) thetransgenic T cell further comprises an expressed chimeric antigen T-cellreceptor or an engineered T-cell receptor; (b) the transgenic T cell isa human transgenic T cell; (c) DNA encoding the CAR and the kynureninaseis integrated into the genome of the cell; and/or (d) wherein the CAR istargeted to a cancer-cell antigen selected from the group consisting ofHER2, CD19, CD20, and GD2.
 20. A method of promoting a T-cell responsein a human subject having a tumor comprising administering an effectiveamount of transgenic cells of claim 19 to the subject.